Application of Cyanobacteria as Chassis Cells in Synthetic Biology.

Synthetic biology is an exciting new area of research that combines science and engineering to design and build new biological functions and systems. Predictably, with the development of synthetic biology, more efficient and economical photosynthetic microalgae chassis will be successfully constructed, making it possible to break through laboratory research into large-scale industrial applications. The synthesis of a range of biochemicals has been demonstrated in cyanobacteria; however, low product titers are the biggest barrier to the commercialization of cyanobacterial biotechnology. This review summarizes the applied improvement strategies from the perspectives of cyanobacteria chassis cells and synthetic biology. The harvest advantages of cyanobacterial products and the latest progress in improving production strategies are discussed according to the product status. As cyanobacteria synthetic biology is still in its infancy, apart from the achievements made, the difficulties and challenges in the application and development of cyanobacteria genetic tool kits in biochemical synthesis, environmental monitoring, and remediation were assessed.

Metabolic engineering for compartmentalized biosynthesis of the valuable compounds in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is commonly used as a microbial cell factory to produce high-value compounds or bulk chemicals due to its genetic operability and suitable intracellular physiological environment. The current biosynthesis pathway for targeted products is primarily rewired in the cytosolic compartment. However, the related precursors, enzymes, and cofactors are frequently distributed in various subcellular compartments, which may limit targeted compounds biosynthesis. To overcome above mentioned limitations, the biosynthesis pathways are localized in different subcellular organelles for product biosynthesis. Subcellular compartmentalization in the production of targeted compounds offers several advantages, mainly relieving competition for precursors from side pathways, improving biosynthesis efficiency in confined spaces, and alleviating the cytotoxicity of certain hydrophobic products. In recent years, subcellular compartmentalization in targeted compound biosynthesis has received extensive attention and has met satisfactory expectations. In this review, we summarize the recent advances in the compartmentalized biosynthesis of the valuable compounds in S. cerevisiae, including terpenoids, sterols, alkaloids, organic acids, and fatty alcohols, etc. Additionally, we describe the characteristics and suitability of different organelles for specific compounds, based on the optimization of pathway reconstruction, cofactor supplementation, and the synthesis of key precursors (metabolites). Finally, we discuss the current challenges and strategies in the field of compartmentalized biosynthesis through subcellular engineering, which will facilitate the production of the complex valuable compounds and offer potential solutions to improve product specificity and productivity in industrial processes.

Alter codon bias of the P. pastoris genome to overcome a bottleneck in codon optimization strategy development and improve protein expression.

Apart from its role in translation, codon bias is also an important mechanism to regulate mRNA levels. The traditional frequency-based codon optimization strategy is rather efficient in organisms such as N. crassa, but much less in yeast P. pastoris which is a popular host for heterologous protein expression. This is because that unlike N. crassa, the preferred codons of P. pastoris are actually AU-rich and hence codon optimization for extremely low GC content comes with issues of pre-mature transcriptional termination or low RNA stability in spite of translational advantages. To overcome this bottleneck, we focused on three reporter genes in P. pastoris first and confirmed the great advantage of GC-prone codon optimization on mRNA levels. Then we altered the codon bias profile of P. pastoris by introducing additional rare tRNA gene copies. Prior to that we constructed IPTG-regulated tRNA species to enable chassis cells to switch between different codon bias status. As demonstrated again with reporter genes, protein yield of luc and 0788 was successfully increased by 4-5 folds in chassis cells. In summary, here we provide an alternative codon optimization strategy for genes with unsatisfactory performance under traditional codon frequency-based optimization.

[Advances in abscisic acid biosynthesis].

Abscisic acid, a plant hormone that inhibits growth, is a key factor in balancing plant endogenous hormones and regulating growth and metabolism. Abscisic acid can improve the drought resistance and salt tolerance of crops, reduce fruit browning, reduce the incidence rate of malaria and stimulate insulin secretion, so it has a broad application potential in agriculture and medicine. Compared with traditional plant extraction and chemical synthesis, abscisic acid synthesis by microorganisms is an economic and sustainable route. At present, a lot of progress has been made in the synthesis of abscisic acid by natural microorganisms such as Botrytis cinerea and Cercospora rosea, while the research on the synthesis of abscisic acid by engineered microorganisms is rarely reported. Saccharomyces cerevisiae, Yarrowia lipolytica and Escherichia coli are common hosts for heterologous synthesis of natural products due to their advantages of clear genetic background, easy operation and friendliness for industrial production. Therefore, the heterologous synthesis of abscisic acid by microorganisms is a more promising production method. The author reviews the research on the heterologous synthesis of abscisic acid by microorganisms from five aspects: selection of chassis cells, screening and expression enhancement of key enzymes, regulation of cofactors, enhancement of precursor supply and promotion of abscisic acid efflux. Finally, the future development direction of this field is prospected.

[Advances in the production of chemicals by organelle compartmentalization in Saccharomyces cerevisiae].

As a generally-recognized-as-safe microorganism, Saccharomyces cerevisiae is a widely studied chassis cell for the production of high-value or bulk chemicals in the field of synthetic biology. In recent years, a large number of synthesis pathways of chemicals have been established and optimized in S. cerevisiae by various metabolic engineering strategies, and the production of some chemicals have shown the potential of commercialization. As a eukaryote, S. cerevisiae has a complete inner membrane system and complex organelle compartments, and these compartments generally have higher concentrations of the precursor substrates (such as acetyl-CoA in mitochondria), or have sufficient enzymes, cofactors and energy which are required for the synthesis of some chemicals. These features may provide a more suitable physical and chemical environment for the biosynthesis of the targeted chemicals. However, the structural features of different organelles hinder the synthesis of specific chemicals. In order to ameliorate the efficiency of product biosynthesis, researchers have carried out a number of targeted modifications to the organelles grounded on an in-depth analysis of the characteristics of different organelles and the suitability of the production of target chemicals biosynthesis pathway to the organelles. In this review, the reconstruction and optimization of the biosynthesis pathways for production of chemicals by organelle mitochondria, peroxisome, golgi apparatus, endoplasmic reticulum, lipid droplets and vacuole compartmentalization in S. cerevisiae are reviewed in-depth. Current difficulties, challenges and future perspectives are highlighted.

Computer-Based Design of a Cell Factory for High-Yield Cytidine Production.

Pyrimidine ribonucleotide de novo biosynthesis pathway (PRdnBP) is an important pathway to produce pyrimidine nucleosides. We attempted to systematically investigate PRdnBP in Escherichia coli with genome-scale metabolic models and utilized the models to guide strain design. The balance of central carbon metabolism and PRdnBP affected the production of cytidine from glucose. Using Bayesian metabolic flux analysis, the effect of modified PRdnBP on the metabolic network was analyzed. The acetate overflow became coupled with PRdnBP flux, while they were originally independent under oxygen-sufficient conditions. The coupling between cytidine production and acetate secretion in the modified strain was weakened by arcA deletion, which resulted in further improving the efficient accumulation of cytidine. In total, 1.28 g/L of cytidine with a yield of 0.26 g/g glucose was produced. The yield of cytidine produced by E. coli is higher than previous reports. Our strategy provides an effective attempt to find metabolic bottlenecks in genetically engineered bacteria by using flux coupling analysis.

Flaviolin-Like Gene Cluster Deletion Optimized the Butenyl-Spinosyn Biosynthesis Route in Saccharopolyspora pogona.

Reduction and optimization of the microbial genome is an important strategy for constructing synthetic biological chassis cells and overcoming obstacles in natural product discovery and production. However, it is of great challenge to discover target genes that can be deleted and optimized due to the complicated genome of actinomycetes. Saccharopolyspora pogona can produce butenyl-spinosyn during aerobic fermentation, and its genome contains 32 different gene clusters. This suggests that there is a large amount of potential competitive metabolism in S. pogona, which affects the biosynthesis of butenyl-spinosyn. By analyzing the genome of S. pogona, six polyketide gene clusters were identified. From those, the complete deletion of clu13, a flaviolin-like gene cluster, generated a high butenyl-spinosyn-producing strain. Production of this strain was 4.06-fold higher than that of the wildtype strain. Transcriptome profiling revealed that butenyl-spinosyn biosynthesis was not primarily induced by the polyketide synthase RppA-like but was related to hypothetical protein Sp1764. However, the repression of sp1764 was not enough to explain the enormous enhancement of butenyl-spinosyn yields in S. pogona-Δclu13. After the comparative proteomic analysis of S. pogona-Δclu13 and S. pogona, two proteins, biotin carboxyl carrier protein (BccA) and response regulator (Reg), were investigated, whose overexpression led to great advantages of butenyl-spinosyn biosynthesis. In this way, we successfully discovered three key genes that obviously optimize the biosynthesis of butenyl-spinosyn. Gene cluster simplification performed in conjunction with multiomics analysis is of great practical significance for screening dominant chassis strains and optimizing secondary metabolism. This work provided an idea about screening key factors and efficient construction of production strains.

Advances in Enhanced Menaquinone-7 Production From Bacillus subtilis.

The production of nutraceutical compounds through biosynthetic approaches has received considerable attention in recent years. For example, Menaquinone-7 (MK-7), a sub-type of Vitamin K2, biosynthesized from Bacillus subtilis (B. subtilis), proved to be more efficiently produced than the conventional chemical synthesis techniques. This is possible due to the development of B. subtilis as a chassis cell during the biosynthesis stages. Hence, it is imperative to provide insights on the B. subtilis membrane permeability modifications, biofilm reactors, and fermentation optimization as advanced techniques relevant to MK-7 production. Although the traditional gene-editing method of homologous recombination improves the biosynthetic pathway, CRISPR-Cas9 could potentially resolve the drawbacks of traditional genome editing techniques. For these reasons, future studies should explore the applications of CRISPRi (CRISPR interference) and CRISPRa (CRISPR activation) system gene-editing tools in the MK-7 anabolism pathway.

[Progress and perspective on development of non-model industrial bacteria as chassis cells for biochemical production in the synthetic biology era].

The development and implement of microbial chassis cells can provide excellent cell factories for diverse industrial applications, which help achieve the goal of environmental protection and sustainable bioeconomy. The synthetic biology strategy of Design-Build-Test-Learn (DBTL) plays a crucial role on rational and/or semi-rational construction or modification of chassis cells to achieve the goals of "Building to Understand" and "Building for Applications". In this review, we briefly comment on the technical development of the DBTL cycle and the research progress of a few model microorganisms. We mainly focuse on non-model bacterial cell factories with potential industrial applications, which possess unique physiological and biochemical characteristics, capabilities of utilizing one-carbon compounds or of producing platform compounds efficiently. We also propose strategies for the efficient and effective construction and application of synthetic microbial cell factories securely in the synthetic biology era, which are to discover and integrate the advantages of model and non-model industrial microorganisms, to develop and deploy intelligent automated equipment for cost-effective high-throughput screening and characterization of chassis cells as well as big-data platforms for storing, retrieving, analyzing, simulating, integrating, and visualizing omics datasets at both molecular and phenotypic levels, so that we can build both high-quality digital cell models and optimized chassis cells to guide the rational design and construction of microbial cell factories for diverse industrial applications.

Fitness of Chassis Cells and Metabolic Pathways for l-Cysteine Overproduction in Escherichia coli.

l-Cysteine is a ubiquitous and unique sulfur-containing amino acid with numerous applications in agricultural and food industries. The efficient production of l-cysteine via microbial fermentation has received a great deal of attention. In this study, the fitness of different Escherichia coli K-12 strains harboring plasmid pLH03 was investigated. The enhancement of the precursor synthetic pathway and thiosulfate assimilation pathway resulted in the good performance of the E. coli BW25113 strain. The expression levels of synthetic pathway genes were optimized by two constitutive promoters to assess their effects on cysteine production. In conjunction, the main degradation pathway genes were also deleted for more efficient production of cysteine. l-Cysteine production was further increased through the manipulation of the sulfur transcription regulator cysB and sulfur supplementation. After process optimization in a 1.5 L bioreactor, LH2A1M0BΔYTS-pLH03 [BW25113 Ptrc2-serA Ptrc1-cysMPtrc-cysBΔyhaMΔtnaAΔsdaA-(pLH03)] accumulated 8.34 g/L cysteine, laying a foundation for application in the cysteine fermentation industry.

Microbial Chassis Development for Natural Product Biosynthesis.

Engineering microbial cells to efficiently synthesize high-value-added natural products has received increasing attention in recent years. In this review, we describe the pipeline to build chassis cells for natural product production. First, we discuss recently developed genome mining strategies for identifying and designing biosynthetic modules and compare the characteristics of different host microbes. Then, we summarize state-of-the-art systems metabolic engineering tools for reconstructing and fine-tuning biosynthetic pathways and transport mechanisms. Finally, we discuss the future prospects of building next-generation chassis cells for the production of natural products. This review provides theoretical guidance for the rational design and construction of microbial strains to produce natural products.

Synthetic Biology Toolbox and Chassis Development in Bacillus subtilis.

Based on technical advances in the sequencing and synthesis of genetic components as well as the genome, significant progress has recently been made in developing synthetic biology toolboxes and chassis for the model Gram-positive bacterium Bacillus subtilis. In this review, we discuss recently developed synthetic biology toolboxes, including gene expression toolsets and genome editing tools. Next, advances in the B. subtilis chassis and its applications are discussed in comparison to those of other model microorganisms. Finally, future directions for the integrative use of B. subtilis synthetic biology tools and the development of an advanced chassis for efficient biomanufacturing are discussed. These factors are expected to become a major driving force for facilitating biotechnological applications of B. subtilis.