Corynebacterium glutamicum cell factory design for the efficient production of cis, cis-muconic acid.

Cis, cis-muconic acid (MA) is widely used as a key starting material in the synthesis of diverse polymers. The growing demand in these industries has led to an increased need for MA. Here, we constructed recombinant Corynebacterium glutamicum by systems metabolic engineering, which exhibit high efficiency in the production of MA. Firstly, the three major degradation pathways were disrupted in the MA production process. Subsequently, metabolic optimization strategies were predicted by computational design and the shikimate pathway was reconstructed, significantly enhancing its metabolic flux. Finally, through optimization and integration of key genes involved in MA production, the recombinant strain produced 88.2 g/L of MA with the yield of 0.30 mol/mol glucose in the 5 L bioreactor. This titer represents the highest reported titer achieved using glucose as the carbon source in current studies, and the yield is the highest reported for MA production from glucose in Corynebacterium glutamicum. Furthermore, to enable the utilization of more cost-effective glucose derived from corn straw hydrolysate, we subjected the strain to adaptive laboratory evolution in corn straw hydrolysate. Ultimately, we successfully achieved MA production in a high solid loading of corn straw hydrolysate (with the glucose concentration of 83.56 g/L), resulting in a titer of 19.9 g/L for MA, which is 4.1 times higher than that of the original strain. Additionally, the glucose yield was improved to 0.33 mol/mol. These provide possibilities for a greener and more sustainable production of MA.

Increased CO2 fixation enables high carbon-yield production of 3-hydroxypropionic acid in yeast.

CO2 fixation plays a key role to make biobased production cost competitive. Here, we use 3-hydroxypropionic acid (3-HP) to showcase how CO2 fixation enables approaching theoretical-yield production. Using genome-scale metabolic models to calculate the production envelope, we demonstrate that the provision of bicarbonate, formed from CO2, restricts previous attempts for high yield production of 3-HP. We thus develop multiple strategies for bicarbonate uptake, including the identification of Sul1 as a potential bicarbonate transporter, domain swapping of malonyl-CoA reductase, identification of Esbp6 as a potential 3-HP exporter, and deletion of Uga1 to prevent 3-HP degradation. The combined rational engineering increases 3-HP production from 0.14 g/L to 11.25 g/L in shake flask using 20 g/L glucose, approaching the maximum theoretical yield with concurrent biomass formation. The engineered yeast forms the basis for commercialization of bio-acrylic acid, while our CO2 fixation strategies pave the way for CO2 being used as the sole carbon source.

Poly(ethylene succinate-co-lactic acid) as a Multifunctional Additive for Modulating the Miscibility, Crystallization, and Mechanical Properties of Poly(lactic acid).

Polymer blending offers an effective and economical approach to overcome the performance limitations of poly(lactic acid) (PLA). In this study, a series of copolymers poly(ethylene succinate-co-lactic acid) (PESL) were synthesized, featuring lactic acid (LA) contents that ranged from 20 to 86 wt %. This synthesis involved a one-pot industrial melt polycondensation process using succinic acid (SA), ethylene glycol (EG), and LA, catalyzed by titanium tetraisopropoxide (TTP). The goal was to produce a fully biobased copolymer expected to exhibit partial miscibility with pure poly(lactic acid) (PLA). To assess the capability of PESL copolymers in toughening PLA, we conducted tensile testing on PLA/PESL blends containing 15 wt % PESL. As a result, an elongation at break for the blends with 15 wt % loading of the copolymer PESL72 was directly enhanced to 250% with an ultimate strength of 35 MPa, compared to brittle PLA with less 10% tensile length. The morphological features of interfacial adhesion before and after tensile failure were measured by scanning electron microscopy (SEM). A significant enhancement in the chain mobility of the PLA/PESL blends was further evidenced by differential scanning calorimetry (DSC) and dynamic mechanical analysis (DMA). These findings hold promise for the development of functional packaging materials based on PLA. The proposed copolymer design, which boasts strong industrial feasibility, can serve as a valuable guide for enhancing the toughness of PLA.

Hydrogel-encapsulated medium chain lipid-modified zeolite imidazole framework-90 as a promising platform for oral delivery of proteins.

The administration of protein therapeutics through oral means is seen as a convenient and painless experience for patients, making it a significant consideration in the field of drug delivery. Nevertheless, the challenging conditions within the gastrointestinal tract, along with the obstacles to absorption, impede the efficient transportation of proteins. Here, we successfully implemented post-synthetic modifications to attach medium-chain lipids (C10) onto the surface of zeolitic imidazole framework-90 (ZIF-90), then encapsulated the nanoparticles with sodium alginate, resulting in a potential platform for the oral administration of proteins. By means of biomimetic mineralization, ZIF-90 achieves a simple and efficient encapsulation of proteins of varying sizes, while shielding them against degradation by digestive enzymes. Sodium alginate hydrogel protects proteins against gastric acid and helps the cargo to rapidly penetrate the mucus layer. Through a mixed mechanism dominated by micropinocytosis, the C10-conjugated ZIF-90 (ZIF-90-C10) can be uptake by Caco-2 cells with a 200-400% increase and transported through the Golgi apparatus after escaping from lysosomes, exhibiting enhanced uptake in the overall gastrointestinal tract. Furthermore, ZIF-90-C10 retains its adenosine triphosphate-responsive release, which drastically lowers the likelihood of accumulation in vivo and allows targeted delivery for disease cells. Our work highlights mid-chain lipid conjugation as a potent approach to enhancing nanoparticle delivery efficiency and a potential strategy for oral delivery of biomacromolecules when combined with pH-responsive gels.

Genome-scale metabolic network models for industrial microorganisms metabolic engineering: Current advances and future prospects.

The construction of high-performance microbial cell factories (MCFs) is the centerpiece of biomanufacturing. However, the complex metabolic regulatory network of microorganisms poses great challenges for the efficient design and construction of MCFs. The genome-scale metabolic network models (GSMs) can systematically simulate the metabolic regulation process of microorganisms in silico, providing effective guidance for the rapid design and construction of MCFs. In this review, we summarized the development status of 16 important industrial microbial GSMs, and further outline the technologies or methods that continuously promote high-quality GSMs construction from five aspects: I) Databases and modeling tools facilitate GSMs reconstruction; II) evolving gap-filling technologies; III) constraint-based model reconstruction; IV) advances in algorithms; and V) developed visualization tools. In addition, we also summarized the applications of GSMs in guiding metabolic engineering from four aspects: I) exploring and explaining metabolic features; II) predicting the effects of genetic perturbations on metabolism; III) predicting the optimal phenotype; IV) guiding cell factories construction in practical experiment. Finally, we discussed the development of GSMs, aiming to provide a reference for efficiently reconstructing GSMs and guiding metabolic engineering.

Sequential catalytic lignin valorization and bioethanol production: an integrated biorefinery strategy.

BACKGROUND: The effective valorization of lignin and carbohydrates in lignocellulose matrix under the concept of biorefinery is a primary strategy to produce sustainable chemicals and fuels. Based on the reductive catalytic fractionation (RCF), lignin in lignocelluloses can be depolymerized into viscous oils, while the highly delignified pulps with high polysaccharides retention can be transformed into various chemicals. RESULTS: A biorefinery paradigm for sequentially valorization of the main components in poplar sawdust was constructed. In this process, the well-defined low-molecular-weight phenols and bioethanol were co-generated by tandem chemo-catalysis in the RCF stage and bio-catalysis in fermentation stage. In the RCF stage, hydrogen transfer reactions were conducted in one-pot process using Raney Ni as catalyst, while the isopropanol (2-PrOH) in the initial liquor was served as a hydrogen donor and the solvent for lignin dissolution. Results indicated the proportion of the 2-PrOH in the initial liquor of RCF influenced the chemical constitution and yield of the lignin oil, which also affected the characteristics of the pulps and the following bioethanol production. A 67.48 ± 0.44% delignification with 20.65 ± 0.31% of monolignols yield were realized when the 2-PrOH:H2O ratio in initial liquor was 7:3 (6.67 wt% of the catalyst loading, 200 °C for 3 h). The RCF pulp had higher carbohydrates retention (57.96 ± 2.78 wt%), which was converted to 21.61 ± 0.62 g/L of bioethanol with a yield of 0.429 ± 0.010 g/g in fermentation using an engineered S. cerevisiae strain. Based on the mass balance analysis, 104.4 g of ethanol and 206.5 g of lignin oil can be produced from 1000 g of the raw poplar sawdust. CONCLUSIONS: The main chemical components in poplar sawdust can be effectively transformed into lignin oil and bioethanol. The attractive results from the biorefinery process exhibit great promise for the production of valuable biofuels and chemicals from abundant lignocellulosic materials.

Surfactant-assisted dilute ethylenediamine fractionation of corn stover for technical lignin valorization and biobutanol production.

In this study, a surfactant-assisted diluted ethylenediamine (EDA) fractionation process was investigated for co-generation of technical lignin and biobutanol from corn stover. The results showed that the addition of PEG 8000 significantly enhanced cellulose recovery (88.9 %) and lignin removal (68.9 %) in the solid fraction. Moreover, the pulp achieved 86.5 % glucose yield and 82.6 % xylose yield in enzymatic hydrolysis. Structural characterization confirmed that the fractionation process promoted the preservation of active ß-O-4 bonds (35.8/100R) in isolated lignin and functionalized the lignin through structural modification using EDA and surfactant grafting. The enzymatic hydrolysate of the pulps yielded a sugar solution for acetone-butanol-ethanol (ABE) fermentation, resulting in an ABE concentration of 15.4 g/L and an overall yield of 137.2 g/Kg of dried corn stalk. Thus, the surfactant-assisted diluted EDA fractionation has the potential to enhance the overall economic feasibility of second-generation biofuels production within the framework of biorefinery.

De novo biosynthesis of 2-hydroxyterephthalic acid, the monomer for high-performance hydroxyl modified PBO fiber, by enzymatic Kolbe-Schmitt reaction with CO2 fixation.

BACKGROUND: High-performance poly(p-phenylenebenzobisoxazole) (PBO) fiber, with excellent mechanical properties (stiffness, strength, and toughness), high thermal stability combined and light weight, are widely employed in automotive and aerospace composites, body armor and sports goods. Hydroxyl modified PBO (HPBO) fiber shows better photostability and interfacial shear strength. 2-Hydroxyterephthalic acid (2-HTA), the monomer for the HPBO fiber, is usually synthesized by chemical method, which has poor space selectivity and high energy consumption. The enzymatic Kolbe-Schmitt reaction, which carboxylates phenolic substrates to generate hydroxybenzoic acids with bicarbonate/CO2, was applied in de novo biosynthesis of 2-HTA with CO2 fixation. RESULTS: The biosynthesis of 2-HTA was achieved by the innovative application of hydroxybenzoic acid (de)carboxylases to carboxylation of 3-hydroxybenzoic acid (3-HBA) at the para-position of the benzene carboxyl group, known as enzymatic Kolbe-Schmitt reaction. 2,3-Dihydroxybenzoic acid decarboxylase from Aspergillus oryzae (2,3-DHBD_Ao) were expressed in recombinant E. coli and showed highest activity. The yield of 2-HTA was 108.97 ± 2.21 µg/L/mg protein in the whole-cell catalysis. In addition, two amino acid substitutions, F27G and T62A, proved to be of great help in improving 2,3-DHBD activity. The double site mutation F27G/T62A increased the production of 2-HTA in the whole-cell catalysis by 24.7-fold, reaching 2.69 ± 0.029 mg/L/mg protein. Moreover, de novo biosynthetic pathway of 2-HTA was constructed by co-expression of 2,3-DHBD_Ao and 3-hydroxybenzoate synthase Hyg5 in S. cerevisiae S288C with Ura3, Aro7 and Trp3 knockout. The engineered strain synthesized 45.40 ± 0.28 µg/L 2-HTA at 36 h in the CO2 environment. CONCLUSIONS: De novo synthesis of 2-HTA has been achieved, using glucose as a raw material to generate shikimic acid, chorismic acid, and 3-HBA, and finally 2-HTA. We demonstrate the strong potential of hydroxybenzoate (de)carboxylase to produce terephthalic acid and its derivatives with CO2 fixation.

Engineering Corynebacterium glutamicum for the efficient production of N-acetylglucosamine.

N-acetylglucosamine (GlcNAc) is significant functional monosaccharides with diverse applications in medicine, food, and cosmetics. In this study, the GlcNAc synthesis pathway was constructed in Corynebacterium glutamicum and its reverse byproduct pathways were blocked. Simultaneously the driving force of GlcNAc synthesis was enhanced by screening key gene sources and inhibiting the GlcNAc consumption pathway. To maximize carbon flux, some competitive pathways (Pentose phosphate pathway, Glycolysis pathway and Mannose pathway) were weakened and the titer of GlcNAc reached 23.30 g/L in shake flasks. Through transcriptome analysis, it was found that dissolved oxygen was an important limiting factor, which was optimized in a 5 L bioreactor. Employing optimal fermentation conditions and feeding strategy, the titer of GlcNAc reached 138.9 g/L, with the yeild of 0.44 g/g glucose. This study significantly increased the yield and titer of GlcNAc, which lay a solid foundation for the industrial production of GlcNAc in C. glutamicum.

Metabolic Engineering for Effective Synthesis of 2-Hydroxyadipate.

Adipic acid is an important monomer in the synthesis of nylon-6,6. In recent years, the biosynthesis of adipic acid has received more and more attention. The pathway with l-lysine as a precursor has potential for adipic acid synthesis, and 2-hydroxyadipate is a key intermediate metabolite in this pathway. In this Letter, the biosynthesis pathway of 2-hydroxyadipate was constructed in Escherichia coli. Through enhancement of precursor synthesis and cofactors regulation, 7.11 g/L of 2-hydroxyadipate was produced in the 5 L bioreactor, which verified the scale-up potential of 2-hydroxyadipate production. Furthermore, 11.1 g/L of 2-hydroxyadipate was produced in the 5 L bioreactor on the basis of potential optimization strategies via transcriptome analysis. This is the first time for the biosynthesis of 2-hydroxyadipate. The results lay a solid foundation for the biosynthesis of adipic acid and the production of bionylon.

A Highly Sensitive Model Based on Graph Neural Networks for Enzyme Key Catalytic Residue Prediction.

Determining the catalytic site of enzymes is a great help for understanding the relationship between protein sequence, structure, and function, which provides the basis and targets for designing, modifying, and enhancing enzyme activity. The unique local spatial configuration bound to the substrate at the active center of the enzyme determines the catalytic ability of enzymes and plays an important role in the catalytic site prediction. As a suitable tool, the graph neural network can better understand and identify the residue sites with unique local spatial configurations due to its remarkable ability to characterize the three-dimensional structural features of proteins. Consequently, a novel model for predicting enzyme catalytic sites has been developed, which incorporates a uniquely designed adaptive edge-gated graph attention neural network (AEGAN). This model is capable of effectively handling sequential and structural characteristics of proteins at various levels, and the extracted features enable an accurate description of the local spatial configuration of the enzyme active site by sampling the local space around candidate residues and special design of amino acid physical and chemical properties. To evaluate its performance, the model was compared with existing catalytic site prediction models using different benchmark datasets and achieved the best results on each benchmark dataset. The model exhibited a sensitivity of 0.9659, accuracy of 0.9226, and area under the precision-recall curve (AUPRC) of 0.9241 on the independent test set constructed for evaluation. Furthermore, the F1-score of this model is nearly four times higher than that of the best-performing similar model in previous studies. This research can serve as a valuable tool to help researchers understand protein sequence-structure-function relationships while facilitating the characterization of novel enzymes of unknown function.

Biosynthesis of 1,3-Propanediol via a New Pathway from Glucose in Escherichia coli.

1,3-Propanediol (1,3-PDO), an important dihydric alcohol, is widely used in textiles, resins, and pharmaceuticals. More importantly, it can be used as a monomer in the synthesis of polytrimethylene terephthalate (PTT). In this study, a new biosynthetic pathway is proposed to produce 1,3-PDO using glucose as a substrate and l-aspartate as a precursor without the addition of expensive vitamin B12. We introduced a 3-HP synthesis module derived from l-aspartate and a 1,3-PDO synthesis module to achieve the de novo biosynthesis. The following strategies were then pursued that included screening key enzymes, optimizing the transcription and translation levels, enhancing the precursor supply of l-aspartate and oxaloacetate, weakening the tricarboxylic acid (TCA) cycle, and blocking competitive pathways. We also used transcriptomic methods to analyze the different gene expression levels. Finally, an engineered Escherichia coli strain produced 6.41 g/L 1,3-PDO with a yield of 0.51 mol/mol glucose in a shake flask and 11.21 g/L in fed-batch fermentation. This study provides a new pathway for production of 1,3-PDO.

Accelerate Large-Scale Biomass Residue Utilization via Cofiring to Help China Achieve Its 2030 Carbon-Peaking Goals.

Cofiring biomass with coal for power generation is an affordable and ready-to-deploy technology to help reduce carbon emissions and resolve residual biomass. Cofiring has not been widely applied in China primarily because of some practical limitations, i.e., biomass accessibility, technological and economic constraints, and lack of policy support. We identified the benefits of cofiring with consideration of these practical limitations based on Integrated Assessment Models. We found that China produces 1.82 Bts/year of biomass residues, 45% of which is waste. 48% of the unused biomass can be utilized without fiscal intervention and 70% can be utilized with the subsidized Feed-in-Tariffs for biopower and carbon trading. The average Marginal Abatement Cost of cofiring is twice that of China's current carbon price. Cofiring can help China create 153 billion yuan of farmers' income annually and reduce 5.3 Bts of Committed Cumulative Carbon Emissions (CCCEs, 2023-2030), contributing to the needed CCCE mitigations to China's overall sector and the power sector by 32 and 86%, respectively. About 201 GW of coal-fired fleets are not compliant with China's 2030 carbon-peaking goals, and 127 GW can be saved by implementing cofiring, representing 9.6% of the total fleets in 2030.

Engineering Escherichia coli BL21 (DE3) for high-yield production of germacrene A, a precursor of ß-elemene via combinatorial metabolic engineering strategies.

ß-elemene is one of the most commonly used antineoplastic drugs in cancer treatment. As a plant-derived natural chemical, biologically engineering microorganisms to produce germacrene A to be converted to ß-elemene harbors great expectations since chemical synthesis and plant isolation methods come with their production deficiencies. In this study, we report the design of an Escherichia coli cell factory for the de novo production of germacrene A to be converted to ß-elemene from a simple carbon source. A series of systematic approaches of engineering the isoprenoid and central carbon pathways, translational and protein engineering of the sesquiterpene synthase, and exporter engineering yielded high-efficient ß-elemene production. Specifically, deleting competing pathways in the central carbon pathway ensured the availability of acetyl-coA, pyruvate, and glyceraldehyde-3-phosphate for the isoprenoid pathways. Adopting lycopene color as a high throughput screening method, an optimized NSY305N was obtained via error-prone polymerase chain reaction mutagenesis. Further overexpression of key pathway enzymes, exporter genes, and translational engineering produced 1161.09 mg/L of ß-elemene in a shake flask. Finally, we detected the highest reported titer of 3.52 g/L of ß-elemene and 2.13 g/L germacrene A produced by an E. coli cell factory in a 4-L fed-batch fermentation. The systematic engineering reported here generally applies to microbial production of a broader range of chemicals. This illustrates that rewiring E. coli central metabolism is viable for producing acetyl-coA-derived and pyruvate-derived molecules cost-effectively.

Enhancing precursor supply and modulating metabolism to achieve high-level production of ß-farnesene in Yarrowia lipolytica.

ß-Farnesene is a sesquiterpene commonly found in essential oils of plants, with applications spanning from agricultural pest control and biofuels to industrial chemicals. The use of renewable substrates in microbial cell factories offers a sustainable approach to ß-farnesene biosynthesis. In this study, malic enzyme from Mucor circinelloides was examined for NADPH regeneration, concomitant with the augmentation of cytosolic acetyl-CoA supply by expressing ATP-citrate lyase from Mus musculus and manipulating the citrate pathway via AMP deaminase and isocitrate dehydrogenase. Carbon flux was modulated through the elimination of native 6-phosphofructokinase, while the incorporation of an exogenous non-oxidative glycolysis pathway served to bridge the pentose phosphate pathway with the mevalonate pathway. The resulting orthogonal precursor supply pathway facilitated ß-farnesene production, reaching 810 mg/L in shake-flask fermentation. Employing optimal fermentation conditions and feeding strategy, a titer of 28.9 g/L of ß-farnesene was attained in a 2 L bioreactor.

Interacting polymer-modification enzymes in heparan sulfate biosynthesis.

Glucuronyl 5-epimerase (Hsepi) converts D-glucuronic acid (GlcA) into L-iduronic acid (IdoA) units, through a mechanism involving reversible abstraction of a proton at C5 of hexuronic acid residues. Incubations of a [4GlcAß1-4GlcNSO3α1-]n precursor substrate with recombinant enzymes in a D2O/H2O medium enabled an isotope exchange approach to the assessment of functional interactions of Hsepi with hexuronyl 2-O-sulfotransferase (Hs2st) and glucosaminyl 6-O-sulfotransferase (Hs6st), both involved in the final polymer-modification steps. Enzyme complexes were supported by computational modeling and homogeneous time resolved fluorescence. GlcA and IdoA D/H ratios related to product composition revealed kinetic isotope effects that were interpreted in terms of efficiency of the coupled epimerase and sulfotransferase reactions. Evidence for a functional Hsepi/Hs6st complex was provided by selective incorporation of D atoms into GlcA units adjacent to 6-O-sulfated glucosamine residues. The inability to achieve simultaneous 2-O- and 6-O-sulfation in vitro supported topologically separated reactions in the cell. These findings provide novel insight into the roles of enzyme interactions in heparan sulfate biosynthesis.

Flux regulation through glycolysis and respiration is balanced by inositol pyrophosphates in yeast.

Although many prokaryotes have glycolysis alternatives, it's considered as the only energy-generating glucose catabolic pathway in eukaryotes. Here, we managed to create a hybrid-glycolysis yeast. Subsequently, we identified an inositol pyrophosphatase encoded by OCA5 that could regulate glycolysis and respiration by adjusting 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP7) levels. 5-InsP7 levels could regulate the expression of genes involved in glycolysis and respiration, representing a global mechanism that could sense ATP levels and regulate central carbon metabolism. The hybrid-glycolysis yeast did not produce ethanol during growth under excess glucose and could produce 2.68 g/L free fatty acids, which is the highest reported production in shake flask of Saccharomyces cerevisiae. This study demonstrated the significance of hybrid-glycolysis yeast and determined Oca5 as an inositol pyrophosphatase controlling the balance between glycolysis and respiration, which may shed light on the role of inositol pyrophosphates in regulating eukaryotic metabolism.

Identification of Emerging Industrial Biotechnology Chassis Vibrio natriegens as a Novel High Salt-Tolerant and Feedstock Flexibility Electroactive Microorganism for Microbial Fuel Cell.

The development of MFC using electroactive industrial microorganisms has seen a surge of interest because of the co-generation for bioproduct and electricity production. Vibrio natriegens as a promising next-generation industrial microorganism chassis and its application for microbial fuel cells (MFC) was first studied. Mediated electron transfer was found in V. natriegens MFC (VMFC), but V. natriegens cannot secrete sufficient electron mediators to transfer electrons to the anode. All seven electron mediators supplemented are capable of improving the electronic transfer efficiency of VMFC. The media and carbon sources switching study reveals that VMFCs have excellent bioelectricity generation performance with feedstock flexibility and high salt-tolerance. Among them, 1% glycerol as the sole carbon source produced the highest power density of 111.9 ± 6.7 mW/cm2. The insight of the endogenous electronic mediators found that phenazine-1-carboxamide, phenazine-1-carboxylic acid, and 1-hydroxyphenazine are synthesized by V. natriegens via the shikimate pathway and the phenazine synthesis and modification pathways. This work provides the first proof for emerging industrial biotechnology chassis V. natriegens as a novel high salt-tolerant and feedstock flexibility electroactive microorganism for MFC, and giving insight into the endogenous electron mediator biosynthesis of VMFC, paving the way for the application of V. natriegens in MFC and even microbial electrofermentation (EF).

Metabolic Engineering Mevalonate Pathway Mediated by RNA Scaffolds for Mevalonate and Isoprene Production in Escherichia coli.

Co-localizing biochemical processes is a great strategy when expressing the heterologous metabolic pathway for product biosynthesis. The RNA scaffold is a flexible and efficient synthetic compartmentalization method to co-localize the enzymes involved in the metabolic pathway by binding to the specific RNA, binding domains fused with the engineered enzymes. Herein, we designed two artificial RNA scaffold structures─0D RNA scaffolds and 2D RNA scaffolds─using the reported aptamers PP7 and BIV-Tat and the corresponding RNA-binding domains (RBDs). We verified the interaction of the RBD and RNA aptamer in vitro and in vivo. Then, we determined the efficiencies of these RNA scaffolds by co-localizing fluorescent proteins. We employed the RNA scaffolds combined with the enzyme fusion strategies to increase the metabolic flux involved in the enzymes of the mevalonate pathway for mevalonate and isoprene production. Compared with the no RNA scaffold strain, the mevalonate levels of the 0D RNA scaffolds and 2D RNA scaffolds increased by 84.1% (3.13 ± 0.03 g/L) and 76.5% (3.00 ± 0.09 g/L), respectively. We applied the 0D RNA scaffolds for increasing the isoprene production by localizing the enzymes involved in a heterologous multi-enzyme pathway. When applying the RNA scaffolds for co-localizing the enzymes mvaE and mvaS, the isoprene production reached to 609.3 ± 57.9 mg/L, increasing by 142% compared with the no RNA scaffold strain. Our results indicate that the RNA scaffold is a powerful tool for improving the efficiencies of the reaction process in the metabolic pathway.

CRISPR-Cas13 technology portfolio and alliance with other genetic tools.

The exploitation of CRISPR-Cas systems especially CRISPR-Cas9 has led to radical advances in genome editing, gene activation, gene repression, protein imaging, and beyond. However, these applications are limited to targeting of DNA rather than RNA. CRISPR-Cas13 is the first reported CRISPR system targeting RNA and thus opens a new avenue for transcription regulation. While a plethora of reviews have systematically documented CRISPR-Cas9 toolbox, this review focuses on CRISPR-Cas13 family, covering aspects of classification, structures, response to foreign invaders, and genetic toolbox. In particular, we compare CRISPR-Cas13 with other RNA regulation tools such as RNA interference and antisense RNA technology to ponder the possibility of combining them to engineer hierarchical regulatory networks fulfilling novel functions. Lastly, we summarize the wide applications of CRISPR-Cas13 toolbox and the status quo that requires amelioration. Overall, this review charts a landscape of CRISPR-Cas13 technology portfolio to provide insights for gene regulation, metabolic engineering and synthetic biology.