Construction of an artificial phosphoketolase pathway that efficiently catabolizes multiple carbon sources to acetyl-CoA.

The canonical glycolysis pathway is responsible for converting glucose into 2 molecules of acetyl-coenzyme A (acetyl-CoA) through a cascade of 11 biochemical reactions. Here, we have designed and constructed an artificial phosphoketolase (APK) pathway, which consists of only 3 types of biochemical reactions. The core enzyme in this pathway is phosphoketolase, while phosphatase and isomerase act as auxiliary enzymes. The APK pathway has the potential to achieve a 100% carbon yield to acetyl-CoA from any monosaccharide by integrating a one-carbon condensation reaction. We tested the APK pathway in vitro, demonstrating that it could efficiently catabolize typical C1-C6 carbohydrates to acetyl-CoA with yields ranging from 83% to 95%. Furthermore, we engineered Escherichia coli stain capable of growth utilizing APK pathway when glycerol act as a carbon source. This novel catabolic pathway holds promising route for future biomanufacturing and offering a stoichiometric production platform using multiple carbon sources.

Rational Design and Modification of NphB for Cannabinoids Biosynthesis.

The rapidly growing field of cannabinoid research is gaining recognition for its impact in neuropsychopharmacology and mood regulation. However, prenyltransferase (NphB) (a key enzyme in cannabinoid precursor synthesis) still needs significant improvement in order to be usable in large-scale industrial applications due to low activity and limited product range. By rational design and high-throughput screening, NphB's catalytic efficiency and product diversity have been markedly enhanced, enabling direct production of a range of cannabinoids, without the need for traditional enzymatic conversions, thus broadening the production scope of cannabinoids, including cannabigerol (CBG), cannabigerolic acid (CBGA), cannabigerovarin (CBGV), and cannabigerovarinic acid (CBGVA). Notably, the W3 mutant achieved a 10.6-fold increase in CBG yield and exhibited a 10.3- and 20.8-fold enhancement in catalytic efficiency for CBGA and CBGV production, respectively. The W4 mutant also displayed an 9.3-fold increase in CBGVA activity. Molecular dynamics simulations revealed that strategic reconfiguration of the active site's hydrogen bonding network, disulfide bond formation, and enhanced hydrophobic interactions are pivotal for the improved synthetic efficiency of these NphB mutants. Our findings advance the understanding of enzyme optimization for cannabinoid synthesis and lay a foundation for the industrial-scale production of these valuable compounds.

Genetically encoded fluorescent sensors reveal dynamic regulation of NADPH metabolism.

Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is essential for biosynthetic reactions and antioxidant functions; however, detection of NADPH metabolism in living cells remains technically challenging. We develop and characterize ratiometric, pH-resistant, genetically encoded fluorescent indicators for NADPH (iNap sensors) with various affinities and wide dynamic range. iNap sensors enabled quantification of cytosolic and mitochondrial NADPH pools that are controlled by cytosolic NAD+ kinase levels and revealed cellular NADPH dynamics under oxidative stress depending on glucose availability. We found that mammalian cells have a strong tendency to maintain physiological NADPH homeostasis, which is regulated by glucose-6-phosphate dehydrogenase and AMP kinase. Moreover, using the iNap sensors we monitor NADPH fluctuations during the activation of macrophage cells or wound response in vivo. These data demonstrate that the iNap sensors will be valuable tools for monitoring NADPH dynamics in live cells and gaining new insights into cell metabolism.

TetraBASE: A Side Chain-Independent Statistical Energy for Designing Realistically Packed Protein Backbones.

To construct backbone structures of high designability is a primary aspect of computational protein design. We report here a side chain-independent statistical energy that aims at realistic modeling of through-space packing of polypeptide backbones. To mitigate the lack of explicit amino acid side chains, the model treats the interbackbone site packing as being dependent on peptide local conformation. In addition, new variables suitable for statistical analysis, one for relative orientation and another for distance, have been introduced to represent the intersite geometry based on the asymmetrical tetrahedron organization of distinct chemical groups surrounding the Cα-carbon atoms. The resulting tetrahedron-based backbone statistical energy (tetraBASE) model has been used to optimize the tertiary organizations of secondary structure elements (SSEs) of designated types with Monte Caro simulated annealing, starting from artificial initial configurations. The tetraBASE minimum energy structures can reproduce SSE packing frequently observed in native proteins with atomic root-mean-square deviations of 1-2 Å. The model has also been tested by examining the stability of native SSE arrangements under tetraBASE. The results suggest that tetraBASE model can be used to effectively represent interbackbone packing when designing backbone structures without explicitly knowing side chain types.

High-Temperature Tolerance Protein Engineering through Deep Evolution.

Protein engineering aimed at increasing temperature tolerance through iterative mutagenesis and high-throughput screening is often labor-intensive. Here, we developed a deep evolution (DeepEvo) strategy to engineer protein high-temperature tolerance by generating and selecting functional sequences using deep learning models. Drawing inspiration from the concept of evolution, we constructed a high-temperature tolerance selector based on a protein language model, acting as selective pressure in the high-dimensional latent spaces of protein sequences to enrich those with high-temperature tolerance. Simultaneously, we developed a variant generator using a generative adversarial network to produce protein sequence variants containing the desired function. Afterward, the iterative process involving the generator and selector was executed to accumulate high-temperature tolerance traits. We experimentally tested this approach on the model protein glyceraldehyde 3-phosphate dehydrogenase, obtaining 8 variants with high-temperature tolerance from just 30 generated sequences, achieving a success rate of over 26%, demonstrating the high efficiency of DeepEvo in engineering protein high-temperature tolerance.

Cytochrome P450 Enzyme Design by Constraining the Catalytic Pocket in a Diffusion Model.

Although cytochrome P450 enzymes are the most versatile biocatalysts in nature, there is insufficient comprehension of the molecular mechanism underlying their functional innovation process. Here, by combining ancestral sequence reconstruction, reverse mutation assay, and progressive forward accumulation, we identified 5 founder residues in the catalytic pocket of flavone 6-hydroxylase (F6H) and proposed a "3-point fixation" model to elucidate the functional innovation mechanisms of P450s in nature. According to this design principle of catalytic pocket, we further developed a de novo diffusion model (P450Diffusion) to generate artificial P450s. Ultimately, among the 17 non-natural P450s we generated, 10 designs exhibited significant F6H activity and 6 exhibited a 1.3- to 3.5-fold increase in catalytic capacity compared to the natural CYP706X1. This work not only explores the design principle of catalytic pockets of P450s, but also provides an insight into the artificial design of P450 enzymes with desired functions.

Origin and evolution of the main starch biosynthetic enzymes.

Starch, a semi-crystalline energy storage form primarily found in plant plastids plays a crucial role in various food or no-food applications. Despite the starch biosynthetic pathway's main enzymes have been characterized, their origin and evolution remained a subject of debate. In this study, we conducted the comprehensive phylogenetic and structural analysis of three types of starch biosynthetic enzymes: starch synthase (SS), starch branching enzyme (SBE) and isoamylase-type debranching enzyme (ISA) from 51,151 annotated genomes. Our findings provide valuable insights into the possible scenario for the origin and evolution of the starch biosynthetic pathway. Initially, the ancestor of SBE can be traced back to an unidentified bacterium that existed before the formation of the last eukaryotic common ancestor (LECA) via horizontal gene transfer (HGT). This transfer event likely provided the eukaryote ancestor with the ability to synthesize glycogen. Furthermore, during the emergence of Archaeplastida, one clade of SS was transferred from Deltaproteobacteria by HGT, while ISA and the other clade of SS originated from Chlamydiae through endosymbiosis gene transfer (EGT). Both these transfer events collectively contributed to the establishment of the original starch biosynthetic pathway. Subsequently, after the divergence of Viridiplantae from Rhodophyta, all three enzymes underwent multiple duplications and N-terminus extension domain modifications, resulting in the formation of functionally specialized isoforms and ultimately leading to the complete starch biosynthetic pathway. By shedding light on the evolutionary origins of key enzymes involved in the starch biosynthetic pathway, this study provides important insights into the evolutionary events of plants.

PCPD: Plant cytochrome P450 database and web-based tools for structural construction and ligand docking.

Plant cytochrome P450s play key roles in the diversification and functional modification of plant natural products. Although over 200,000 plant P450 gene sequences have been recorded, only seven crystalized P450 genes severely hampered the functional characterization, gene mining and engineering of important P450s. Here, we combined Rosetta homologous modeling and MD-based refinement to construct a high-resolution P450 structure prediction process (PCPCM), which was applied to 181 plant P450s with identified functions. Furthermore, we constructed a ligand docking process (PCPLD) that can be applied for plant P450s virtual screening. 10 examples of virtual screening indicated the process can reduce about 80% screening space for next experimental verification. Finally, we constructed a plant P450 database (PCPD: http://p450.biodesign.ac.cn/), which includes the sequences, structures and functions of the 181 plant P450s, and a web service based on PCPCM and PCPLD. Our study not only developed methods for the P450-specific structure analysis, but also introduced a universal approach that can assist the mining and functional analysis of P450 enzymes.

Cell-free chemoenzymatic starch synthesis from carbon dioxide.

Starches, a storage form of carbohydrates, are a major source of calories in the human diet and a primary feedstock for bioindustry. We report a chemical-biochemical hybrid pathway for starch synthesis from carbon dioxide (CO2) and hydrogen in a cell-free system. The artificial starch anabolic pathway (ASAP), consisting of 11 core reactions, was drafted by computational pathway design, established through modular assembly and substitution, and optimized by protein engineering of three bottleneck-associated enzymes. In a chemoenzymatic system with spatial and temporal segregation, ASAP, driven by hydrogen, converts CO2 to starch at a rate of 22 nanomoles of CO2 per minute per milligram of total catalyst, an ~8.5-fold higher rate than starch synthesis in maize. This approach opens the way toward future chemo-biohybrid starch synthesis from CO2.

Chromosome-level genome of Himalayan yew provides insights into the origin and evolution of the paclitaxel biosynthetic pathway.

Taxus, commonly known as yew, is a well-known gymnosperm with great ornamental and medicinal value. In this study, by assembling a chromosome-level genome of the Himalayan yew (Taxus wallichiana) with 10.9 Gb in 12 chromosomes, we revealed that tandem duplication acts as the driving force of gene family evolution in the yew genome, resulting in the main genes for paclitaxel biosynthesis, i.e. those encoding the taxadiene synthase, P450s, and transferases, being clustered on the same chromosome. The tandem duplication may also provide genetic resources for the nature to sculpt the core structure of taxoids at different positions and subsequently establish the complex pathway of paclitaxel by neofunctionalization. Furthermore, we confirmed that there are two genes in the cluster encoding isoenzymes of a known enzyme in the paclitaxel biosynthetic pathway. The reference genome of the Himalayan yew will serve as a platform for decoding the complete biosynthetic pathway of paclitaxel and understanding the chemodiversity of taxoids in gymnosperms.

A functional antibody cross-reactive to both human and murine cytotoxic T-lymphocyte-associated protein 4 via binding to an N-glycosylation epitope.

Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, CD152) is a receptor on T cells that inhibits the cell's functions. Blocking CTLA-4 with an antibody has proven effective for the treatment of cancer patients. Anti-CTLA-4 antibodies currently approved for clinical use can bind to human CTLA-4, but do not cross-react to murine CTLA-4. Here, we report the generation and characterization of a functional humanized antibody, mAb146, against both human and murine CTLA-4. Alanine scanning of CTLA-4 using mammalian cell expression cassette identified the unique epitopes of this novel antibody. In addition to the amino acid residues interacting with ligands CD80 and CD86, an N-glycosylation site on N110, conserved in CTLA-4 of human, monkey, and mouse, was identified as the specific epitope that might contribute to the cross-species binding and function of this antibody. This finding may also contribute to the understanding of the glycosylation of CTLA-4 and its related biologic function. In addition to facilitating preclinical development of anti-CTLA-4 antibodies, mAb146 may be useful as a therapeutic agent.

Structure of the intact ATM/Tel1 kinase.

The ataxia-telangiectasia mutated (ATM) protein is an apical kinase that orchestrates the multifaceted DNA-damage response. Normally, ATM kinase is in an inactive, homodimer form and is transformed into monomers upon activation. Besides a conserved kinase domain at the C terminus, ATM contains three other structural modules, referred to as FAT, FATC and N-terminal helical solenoid. Here we report the first cryo-EM structure of ATM kinase, which is an intact homodimeric ATM/Tel1 from Schizosaccharomyces pombe. We show that two monomers directly contact head-to-head through the FAT and kinase domains. The tandem N-terminal helical solenoid tightly packs against the FAT and kinase domains. The structure suggests that ATM/Tel1 dimer interface and the consecutive HEAT repeats inhibit the binding of kinase substrates and regulators by steric hindrance. Our study provides a structural framework for understanding the mechanisms of ATM/Tel1 regulation as well as the development of new therapeutic agents.