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Abstract Fluorescence lifetime imaging microscopy (FLIM) opens new dimensions for highly multiplexed imaging in live cells and organisms using differences in fluorescence lifetime to distinguish spectrally identical fluorescent probes. Here, a set of fluorescence‐activating and absorption‐shifting tags (FASTs) capable of modulating the fluorescence lifetime of embedded fluorogenic 4‐hydroxybenzylidene rhodanine (HBR) derivatives is described. It is shown that changes in the FAST protein sequence can vary the local environment of the chromophore and lead to significant changes in fluorescence lifetime. These fluorescence lifetime‐modulating tags enable multiplexed imaging of up to three targets in one spectral channel using a single HBR derivative in live cells and live zebrafish larvae. The combination of fluorescence lifetime multiplexing with spectral multiplexing allows to successfully image six targets in live cells, opening great prospects for multicolor fluorescence lifetime multiplexing.

Abstract Synucleinopathies, including Parkinson's disease (PD), multiple system atrophy (MSA), and dementia with Lewy bodies (DLB), are neurodegenerative disorders caused by the accumulation of misfolded alpha‐synuclein protein. Developing effective vaccines against synucleinopathies is challenging due to the difficulty of stimulating an immune‐specific response against alpha‐synuclein without causing harmful autoimmune reactions, selectively targeting only pathological forms of alpha‐synuclein. Previous attempts using linear peptides and epitopes without control of the antigen structure failed in clinical trials. The immune system was unable to distinguish between native alpha‐synuclein and its amyloid form. The prion domain of the fungal HET‐s protein was selected as a scaffold to introduce select epitopes from the surface of alpha‐synuclein fibrils. Four vaccine candidates were generated by introducing specific amino acid substitutions onto the surface of the scaffold protein. The approach successfully mimicked the stacking of the parallel in‐register beta‐sheet structure seen in alpha‐synuclein fibrils. All vaccine candidates induced substantial levels of IgG antibodies that recognized pathological alpha‐synuclein fibrils derived from a synucleinopathy mouse model. Furthermore, the antisera recognized pathological alpha‐synuclein aggregates in brain lysates from patients who died from DLB, MSA, or PD, but did not recognize linear alpha‐synuclein peptides. Our approach, based on the rational design of vaccines using the structure of alpha‐synuclein amyloid fibrils and strict control over the exposed antigen structure used for immunization, as well as the ability to mimic aggregated alpha‐synuclein, provides a promising avenue toward developing effective vaccines against alpha‐synuclein fibrils.

Abstract Acid‐induced arginine decarboxylase AdiA is a typical homo‐oligomeric protein biosynthesizing alkaline nylon monomer putrescine. However, upon loss of the AdiA decamer oligomeric state at neutral and alkaline conditions the activity also diminishes, obstructing the whole‐cell biosynthesis of alkaline putrescine. Here, a structure cohesion strategy is proposed to change the pH adaptation of AdiA to alkaline environments based on the rational engineering of meridional and latitudinal oligomerization interfaces. After integrating substitutions of E467K at the latitudinal interface and H736E at the meridional channel interface, the structural stability of AdiA decamer and its substrate transport efficiency at neutral and alkaline conditions are improved. Finally, E467K_H736E is well adapted to neutral and alkaline environments (pH 7.0–9.0), and its enzymatic activity is 35‐fold higher than that of wild AdiA at pH 8.0. Using E467K_H736E in the putrescine synthesis pathway, the titer of putrescine is up to 128.9 g·L−1 with a conversion of 0.94 mol·mol−1 in whole‐cell catalysis. Additionally, the neutral pH adaptation of lysine decarboxylase, with a decamer structure similar to AdiA, is also improved using this cohesion strategy, providing an option for pH‐adaptation engineering of other oligomeric decarboxylases.

Abstract Genome Editing is widely used in biomedical research and medicine. Zinc finger nucleases (ZFNs) are smaller in size than transcription activator‐like effector (TALE) nucleases (TALENs) and CRISPR‐Cas9. Therefore, ZFN‐encoding DNAs can be easily packaged into a viral vector with limited cargo space, such as adeno‐associated virus (AAV) vectors, for in vivo and clinical applications. ZFNs have great potential for translational research and clinical use. However, constructing functional ZFNs and improving their genome editing efficiency is extremely difficult. Here, the efficient construction of functional ZFNs and the improvement of their genome editing efficiency using AlphaFold, Coot, and Rosetta are described. Plasmids encoding ZFNs consisting of six fingers using publicly available zinc‐finger resources are assembled. Two functional ZFNs from the ten ZFNs tested are successfully obtained. Furthermore, the engineering of ZFNs using AlphaFold, Coot, or Rosetta increases the efficiency of genome editing by 5%, demonstrating the effectiveness of engineering ZFNs based on structural modeling.

Abstract Biosynthesis is the application of enzymes in microbial cell factories and has emerged as a promising alternative to chemical synthesis. However, natural enzymes with limited catalytic performance often need to be engineered to meet specific needs through a time‐consuming trial‐and‐error process. This study presents a quantum mechanics (QM)‐incorporated design–build–test–learn (DBTL) framework to rationally design phosphatase BT4131, an enzyme with an ambiguous substrate spectrum involved in N‐acetylglucosamine (GlcNAc) biosynthesis. First, mutant M1 (L129Q) is designed using force field‐based methods, resulting in a 1.4‐fold increase in substrate preference (kcat/Km) toward GlcNAc‐6‐phosphate (GlcNAc6P). QM calculations indicate that the shift in substrate preference is caused by a 13.59 kcal mol−1 reduction in activation energy. Furthermore, an iterative computer‐aided design is conducted to stabilize the transition state. As a result, mutant M4 (I49Q/L129Q/G172L) with a 9.5‐fold increase in kcat‐GlcNAc6P/Km‐GlcNAc6P and a 59% decrease in kcat‐Glc6P/Km‐Glc6P is highly desirable compared to the wild type in the GlcNAc‐producing chassis. The GlcNAc titer increases to 217.3 g L−1 with a yield of 0.597 g (g glucose)−1 in a 50‐L bioreactor, representing the highest reported level. Collectively, this DBTL framework provides an easy yet fascinating approach to the rational design of enzymes for industrially viable biocatalysts.

Abstract Fast screening strategies that enable high‐throughput evaluation and identification of desired variants from diversified enzyme libraries are crucial to tailoring biocatalysts for the synthesis of D‐allulose, which is currently limited by the poor catalytic performance of ketose 3‐epimerases (KEases). Here, the study designs a minimally equipment‐dependent, high‐throughput, and growth‐coupled in vivo screening platform founded on a redesigned D‐allulose‐dependent biosensor system. The genetic elements modulating regulator PsiR expression levels undergo systematic optimization to improve the growth‐responsive dynamic range of the biosensor, which presents ≈30‐fold facilitated growth optical density with a high signal‐to‐noise ratio (1.52 to 0.05) toward D‐allulose concentrations from 0 to 100 mm. Structural analysis and evolutionary conservation analysis of Agrobacterium sp. SUL3 D‐allulose 3‐epimerase (ADAE) reveal a highly conserved catalytic active site and variable hydrophobic pocket, which together regulate substrate recognition. Structure‐guided rational design and directed evolution are implemented using the growth‐coupled in vivo screening platform to reprogram ADAE, in which a mutant M42 (P38N/V102A/Y201L/S207N/I251R) is identified with a 6.28‐fold enhancement of catalytic activity and significantly improved thermostability with a 2.5‐fold increase of the half‐life at 60 °C. The research demonstrates that biosensor‐assisted growth‐coupled evolutionary pressure combined with structure‐guided rational design provides a universal route for engineering KEases.

Abstract Intravitreal (IVT) injection of anti‐vascular endothelial growth factor (anti‐VEGF) has greatly improved the treatment of many retinal disorders, including wet age‐related macular degeneration (wAMD), which is the third leading cause of blindness. However, frequent injections can be difficult for patients and may lead to various risks such as elevated intraocular pressure, infection, and retinal detachment. To address this issue, researchers have found that IVT injection of anti‐VEGF proteins at their maximally viable concentration and dose can be an effective strategy. However, the intrinsic protein structure can limit the maximum concentration due to stability and solution viscosity. To overcome this challenge, we developed a novel anti‐VEGF protein called nanoFc by fusing anti‐VEGF nanobodies with a crystallizable fragment (Fc). NanoFc has demonstrated high binding affinity to VEGF165 through multivalency and potent bioactivity in various bioassays. Furthermore, nanoFc maintains satisfactory chemical and physical stability at 4°C over 1 month and is easily injectable at concentrations up to 200 mg/mL due to its unique architecture that yields a smaller shape factor. The design of nanoFc offers a bioengineering strategy to ensure both strong anti‐VEGF binding affinity and high protein concentration, with the goal of reducing the frequency of IV injections.

Genetically encoded calcium indicators based on truncated troponin C are attractive probes for calcium imaging due to their relatively small molecular size and twofold reduced calcium ion buffering. However, the best‐suited members of this family, YTnC and cNTnC, suffer from low molecular brightness, limited dynamic range, and/or poor sensitivity to calcium transients in neurons. To overcome these limitations, we developed an enhanced version of YTnC, named YTnC2. Compared with YTnC, YTnC2 had 5.7‐fold higher molecular brightness and 6.4‐fold increased dynamic range in vitro. YTnC2 was successfully used to reveal calcium transients in the cytosol and in the lumen of mitochondria of both mammalian cells and cultured neurons. Finally, we obtained and analyzed the crystal structure of the fluorescent domain of the YTnC2 mutant.

Improving protein stability is important for industrial applications, and one promising method for achieving this is backbone circularization. As connector length affects stability, predicting and elucidating a more stable connector length is necessary for development of the backbone circularization method. However, the relationship between connector length and protein stability has not been completely elucidated. Here, we determined the most stable connector length for granulocyte colony‐stimulating factor by changing one residue at a time to produce connector length variants and then measuring their thermal stability. Analysis of the local structures obtained from the predicted structures of the circularized variants revealed that an approach using helix length, dihedral backbone angle, and number of unbonded hydrogen bond donors and acceptors is suitable for identifying connector lengths with higher stability.

Abstract Dual targeting to immune checkpoints has achieved a better therapeutic efficacy than single targeting due to synergistic extrication of tumour immunity. However, most dual targeting strategies are usually antibody dependent which facing drawbacks of antibodies, such as poor solid tumour penetration and unsatisfied affinity. To meet the challenges, we engineered a cell membrane displaying a fusion protein composed of SIRPα and PD‐1 variants, the high‐affinity consensus (HAC) of wild‐type molecules, and with which prepared nanovesicles (NVs). Through disabling both SIRPα/CD47 and PD‐1/PD‐L1 signalling, HAC NVs significantly preserved the phagocytosis and antitumour effect of macrophages and T cells, respectively. In vivo study revealed that HAC NVs had better tumour penetration than monoclonal antibodies and higher binding affinity to CD47 and PD‐L1 on tumour cells compared with the NVs expressing wild‐type fusion protein. Exhilaratingly, dual‐blockade of CD47 and PD‐L1 with HAC NVs exhibited excellent therapeutic efficacy and biosafety. This study provided a novel biomaterial against tumoural immune escape and more importantly an attractive biomimetic technology of protein delivery for multi‐targeting therapies.

Abstract The cytokine interleukin (IL)‐11 has been shown to play a role in promoting fibrosis and cancer, including lung adenocarcinoma, garnering interest as an attractive target for therapeutic intervention. We used combinatorial methods to engineer an IL‐11 variant that binds with higher affinity to the IL‐11 receptor and stimulates enhanced receptor‐mediated cell signaling. Introduction of two additional point mutations ablates IL‐11 ligand/receptor association with the gp130 coreceptor signaling complex, resulting in a high‐affinity receptor antagonist. Unlike wild‐type IL‐11, this engineered variant potently blocks IL‐11‐mediated cell signaling and slows tumor growth in a mouse model of lung cancer. Our approach highlights a strategy where native ligands can be engineered and exploited to create potent receptor antagonists.

Abstract Domain insertion engineering is a promising approach to recombine the functions of evolutionarily unrelated proteins. Insertion of light‐switchable receptor domains into a selected effector protein, for instance, can yield allosteric effectors with light‐dependent activity. However, the parameters that determine domain insertion tolerance and allostery are poorly understood. Here, an unbiased screen is used to systematically assess the domain insertion permissibility of several evolutionary unrelated proteins. Training machine learning models on the resulting data allow to dissect features informative for domain insertion tolerance and revealed sequence conservation statistics as the strongest indicators of suitable insertion sites. Finally, extending the experimental pipeline toward the identification of switchable hybrids results in opto‐chemogenetic derivatives of the transcription factor AraC that function as single‐protein Boolean logic gates. The study reveals determinants of domain insertion tolerance and yielded multimodally switchable proteins with unique functional properties.

Abstract Antigen delivery based on non‐virus‐like particle self‐associating protein nanoscffolds, such as Aquifex aeolicus lumazine synthase (AaLS), is limited due to the immunotoxicity and/or premature clearance of antigen‐scaffold complex resulted from triggering unregulated innate immune responses. Here, using rational immunoinformatics prediction and computational modeling, we screen the T epitope peptides from thermophilic nanoproteins with the same spatial structure as hyperthermophilic icosahedral AaLS, and reassemble them into a novel thermostable self‐assembling nanoscaffold RPT that can specifically activate T cell‐mediated immunity. Tumor model antigen ovalbumin T epitopes and the severe acute respiratory syndrome coronavirus 2 receptor‐binding domain are loaded onto the scaffold surface through the SpyCather/SpyTag system to construct nanovaccines. Compared to AaLS, RPT‐constructed nanovaccines elicit more potent cytotoxic T cell and CD4+ T helper 1 (Th1)‐biased immune responses, and generate less anti‐scaffold antibody. Moreover, RPT significantly upregulate the expression of transcription factors and cytokines related to the differentiation of type‐1 conventional dendritic cells, promoting the cross‐presentation of antigens to CD8+ T cells and Th1 polarization of CD4+ T cells. RPT confers antigens with increased stability against heating, freeze‐thawing, and lyophilization with almost no antigenicity loss. This novel nanoscaffold offers a simple, safe, and robust strategy for boosting T‐cell immunity‐dependent vaccine development.

Abstract The introduction of heterologous pathways into microorganisms for high‐level synthesis of valuable compounds would be extremely meaningful. However, the endogenous heterologous pathway conversion point frequently serves as a bottleneck that limits product synthesis. (+)‐Nootkatone is an important functional sesquiterpenoid with a unique grapefruit flavor, which is frequently used as a fragrance. The low catalytic efficiency of the valencene synthase CnVS, the first heterologous enzyme in the biosynthesis of (+)‐nootkatone, is the major challenge that restricts the production of (+)‐nootkatone in Saccharomyces cerevisiae. In this study, we modified CnVS on the basis of the catalytic environment and mechanism of the enzyme. The residue M560 involving in alkyl transfer during substrate cyclization was found to affect activity. By tuning the residue, mutant M560L exhibited a 60% increase in the unit cell yield of (+)‐valencene, which is the precursor of (+)‐nootkatone. Furthermore, multistep strategies of metabolic engineering were employed that transferred the metabolic flux to the target product synthesis pathway and displayed the superimposed effect of metabolic and protein engineering on (+)‐nootkatone yield. Finally, multicopy integration of tHMG1 and ERG20ERG20‐GSG‐CnVSM560L boosted the production of (+)‐nootkatone to 85.43 mg/L in flask and 804.96 mg/L (5.29 mg/[L h]) in bioreactor. In conclusion, this study presents a paradigm for constructing microbial cell factories using a combination of protein and metabolic engineering in S. cerevisiae.

Catechol 1,2 dioxygenase is a versatile enzyme with several potential applications. However, due to its low thermostability, its industrial potential is not being met. In this study, the thermostability of a mesophilic catechol 1,2 dioxygenase from the species Rhodococcus opacus was enhanced via the introduction of disulphide bonds into its structure. Engineered designs (56) were obtained using computational prediction applications, with a set of hypothesized selection criteria narrowing the list to 9. Following recombinant production and purification, several of the designs demonstrated substantially improved protein thermostability. Notably, variant K96C-D278C yielded improvements including a 4.6°C increase in T 50 , a 725% increase in half-life, a 5.5°C increase in T m , and a >10-fold increase in total turnover number compared to wild type. Stacking of best designs was not productive. Overall, current state-of-the-art prediction algorithms were effective for design of disulfide-thermostabilized catechol 1,2 dioxygenase., (© 2024 National Research Council Canada and The Authors. Biotechnology and Bioengineering by Wiley Periodicals LLC. Reproduced with the permission of the Minister of Innovation, Science, and Economic Development.)

To reduce carbon emissions and address environmental concerns, the aviation industry is exploring the use of sustainable aviation fuel (SAF) as an alternative to traditional fossil fuels. In this context, bio-alkane is considered a potentially high-value solution. The present study focuses on the enzymes acyl-acyl carrier protein [ACP] reductase (AAR) and aldehyde-deformylating oxygenase (ADO), which are crucial enzymes for alka(e)ne biosynthesis. By using protein engineering techniques, including semi-rational design and site-directed mutagenesis, we aimed to enhance the substrate specificity of AAR and improve alkane production efficiency. The co-expression of a modified AAR (Y26G/Q40M mutant) with wild-type ADO in Escherichia coli significantly increased alka(e)ne production from 28.92 mg/L to 167.30 mg/L, thus notably demonstrating a 36-fold increase in alkane yield. This research highlights the potential of protein engineering in optimizing SAF production, thereby contributing to the development of more sustainable and efficient SAF production methods and promoting greener air travel., (© 2024 Wiley Periodicals LLC.)

Urokinase-type plasminogen activator receptor (uPAR) is overexpressed on tumor cells in multiple types of cancer and contributes to disease progression and metastasis. In this work, we engineered a novel bi-paratopic uPAR targeting agent by fusing the binding domains of two native uPAR ligands: uPA and vitronectin, with a flexible peptide linker. The linker length was optimized to facilitate simultaneous engagement of both domains to their adjacent epitopes on uPAR, resulting in a high affinity and avid binding interaction. Furthermore, the individual domains were affinity-matured using yeast surface display and directed evolution, resulting in a bi-paratopic protein with affinity in the picomolar to femtomolar range. This engineered uPAR targeting agent demonstrated significantly enhanced tumor localization in mouse tumor models compared to the native uPAR ligand and warrants further investigation as a diagnostic and therapeutic agent for cancer., (© 2024 Wiley Periodicals LLC.)

D-amino acid oxidase (DAAO)-catalyzed selective oxidative deamination is a very promising process for synthesizing l-amino acids including l-phosphinothricin (l-PPT, a high-efficiency and broad-spectrum herbicide). However, the wild-type DAAO's low activity toward unnatural substrates like d-phosphinothricin (d-PPT) hampers its application. Herein, a DAAO from Caenorhabditis elegans (CeDAAO) was screened and engineered to improve the catalytic potential on d-PPT. First, we designed a novel growth selection system, taking into account the intricate relationship between the growth of Escherichia coli (E. coli) and the catalytic mechanism of DAAO. The developed system was used for high-throughput screening of gene libraries, resulting in the discovery of a variant (M6) with significantly increased catalytic activity against d-PPT. The variant displays different catalytic properties on substrates with varying hydrophobicity and hydrophilicity. Analysis using Alphafold2 modeling and molecular dynamic simulations showed that the reason for the enhanced activity was the substrate-binding pocket with enlarged size and suitable charge distribution. Further QM/MM calculations revealed that the crucial factor for enhancing activity lies in reducing the initial energy barrier of the reductive half reaction. Finally, a comprehensive binding-model index to predict the enhanced activity of DAAO toward d-PPT, and an enzymatic deracemization approach was developed, enabling the efficient synthesis of l-PPT with remarkable efficiency., (© 2024 Wiley Periodicals LLC.)

Abstract Engineered proteases are promising tools to address physiological and pathophysiological questions as well as to develop new therapeutic approaches. Here we introduce a new genetically encoded engineered single‐chain tobacco etch virus protease, allowing to control proprotein cleavage in different compartments of living mammalian cells. We demonstrated a set of controllable proteolytic effects, including cytosolic protein cleavage, inducible gene expression, and maturation of brain‐derived neurotrophic factor (BDNF) in the secretory pathway thus showing the versatility of this technique. Of note, the secretory pathway exhibits different characteristics from the cytosol and it is difficult to target because inaccessible to some small molecules. We were able to induce ligand‐mediated BDNF maturation and monitor its effects on dendritic spines in hippocampal pyramidal cells and in the mouse brain. This strategy paves the way to dissect proteolytic cleavage product signaling in various processes as well as for future therapeutic applications.

Harnessing DNA as a high-density storage medium for information storage and molecular recording of signals has been of increasing interest in the biotechnology field. Recently, progress in enzymatic DNA synthesis, DNA digital data storage, and DNA-based molecular recording has been made by leveraging the activity of the template-independent DNA polymerase, terminal deoxynucleotidyl transferase (TdT). TdT adds deoxyribonucleotides to the 3' end of single-stranded DNA, generating random sequences of single-stranded DNA. TdT can use several divalent cations for its enzymatic activity and exhibits shifts in deoxyribonucleotide incorporation frequencies in response to changes in its reaction environment. However, there is limited understanding of sequence-structure-function relationships regarding these properties, which in turn limits our ability to modulate TdT to further advance TdT-based tools. Most TdT literature to-date explores the activity of murine, bovine or human TdTs; studies probing TdT sequence and structure largely focus on strictly conserved residues that are functionally critical to TdT activity. Here, we explore non-conserved TdT sequence space by surveying the natural diversity of TdT. We characterize a diverse set of TdT homologs from different organisms and identify several TdT residues/regions that confer differences in TdT behavior between homologs. The observations in this study can design rules for targeted TdT libraries, in tandem with a screening assay, to modulate TdT properties. Moreover, the data can be useful in guiding further studies of potential residues of interest. Overall, we characterize TdTs that have not been previously studied in the literature, and we provide new insights into TdT sequence-function relationships., (© 2024 The Author(s). Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Abstract Protein design plays an important role in recent medical advances from antibody therapy to vaccine design. Typically, exhaustive mutational screens or directed evolution experiments are used for the identification of the best design or for improvements to the wild‐type variant. Even with a high‐throughput screening on pooled libraries and Next‐Generation Sequencing to boost the scale of read‐outs, surveying all the variants with combinatorial mutations for their empirical fitness scores is still of magnitudes beyond the capacity of existing experimental settings. To tackle this challenge, in‐silico approaches using machine learning to predict the fitness of novel variants based on a subset of empirical measurements are now employed. These machine learning models turn out to be useful in many cases, with the premise that the experimentally determined fitness scores and the amino‐acid descriptors of the models are informative. The machine learning models can guide the search for the highest fitness variants, resolve complex epistatic relationships, and highlight bio‐physical rules for protein folding. Using machine learning‐guided approaches, researchers can build more focused libraries, thus relieving themselves from labor‐intensive screens and fast‐tracking the optimization process. Here, we describe the current advances in massive‐scale variant screens, and how machine learning and mutagenesis strategies can be integrated to accelerate protein engineering. More specifically, we examine strategies to make screens more economical, informative, and effective in discovery of useful variants.

Abstract Understanding protein structure and function relationships in cellulose synthase (CesA), including divergent isomers, is an important goal. Here, we report results from mutant complementation assays that tested the ability of sequence variants of AtCesA7, a secondary wall CesA of Arabidopsis thaliana, to rescue the collapsed vessels, short stems, and low cellulose content of the irx3‐1 AtCesA7 null mutant. We tested a catalytic null mutation and seven missense or small domain changes in and near the AtCesA7 FTVTSK motif, which lies near the catalytic domain and may, analogously to bacterial CesA, exist within a substrate “gating loop.” A low‐to‐high gradient of rescue occurred, and even inactive AtCesA7 had a small positive effect on stem cellulose content but not stem elongation. Overall, secondary wall cellulose content and stem length were moderately correlated, but the results were consistent with threshold amounts of cellulose supporting particular developmental processes. Vibrational sum frequency generation microscopy allowed tissue‐specific analysis of cellulose content in stem xylem and interfascicular fibers, revealing subtle differences between selected genotypes that correlated with the extent of rescue of the collapsing xylem phenotype. Similar tests on PpCesA5 from the moss Physcomitrium (formerly Physcomitrella) patens helped us to synergize the AtCesA7 results with prior results on AtCesA1 and PpCesA5. The cumulative results show that the FTVTxK region is important for the function of an angiosperm secondary wall CesA as well as widely divergent primary wall CesAs, while differences in complementation results between isomers may reflect functional differences that can be explored in further work.

Carbonyl reductases are useful for producing optically active alcohols from their corresponding prochiral ketones. Herein, we applied a computer-assisted strategy to increase the thermostability of a previously constructed carbonyl reductase, LsCR M4 (N101D/A117G/F147L/E145A), which showed an outstanding activity in the synthesis of the ticagrelor precursor (1S)-2-chloro-1-(3,4-difluorophenyl)ethanol. The stability changes introduced by mutations at the flexible sites were predicted using the computational tools FoldX, I-Mutant 3.0, and DeepDDG, which demonstrated that 12 virtually screened mutants could be thermally stable; 11 of these mutants exhibited increased thermostability. Then a superior mutant LsCR M4 -V99L/D150F was screened out from the library that was constructed by iteratively combining the beneficial sites, which showed a 78% increase in activity and a 17.4°C increase in melting temperature compared to LsCR M4 . Our computer-assisted design and combinatorial strategy dramatically increased the efficiency of thermostable enzyme production., (© 2024 Wiley Periodicals LLC.)

Bioproduction of chiral amines is limited by low transaminase (TA) activity on nonnatural substrates, leading to the need for protein engineering. To address the challenge of quickly and precisely identifying the engineering targets, a strategy was proposed based on analyzing the mode changes in the high-energy intermediate state (H-state) of the substrate-enzyme complex during catalysis. By substituting the residues with minimal structural changes in catalytically active mode (A-mode) and distance-free mode (F-mode) of the H-state complex with more conserved ones to stabilize it, a TA mutant M5(T295C/L387A/V436A) with 121.9-fold higher activity for synthesizing the (S)-Rivastigmine precursor (S)-1-(3-methoxyphenyl)ethylamine was created. The applicability of this strategy was also validated by engineering another TA for 1.52-fold higher activity and >99% selectivity toward (R)-3-amino-1-butanol biopreparation. The much higher stereoselectivity of the mutant compared with the wild type (28.3%) demonstrated that this strategy is not only advantageous in engineering enzyme activity but also applicable for modulating stereoselectivity., (© 2023 Wiley Periodicals LLC.)

Abstract Nanotechnologies are considered to be of growing importance to the vaccine field. Through decoration of immunogens on multivalent nanoparticles, designed nanovaccines can elicit improved humoral immunity. However, significant practical and monetary challenges in large‐scale production of nanovaccines have impeded their widespread clinical translation. Here, an alternative approach is illustrated integrating computational protein modeling and adaptive electroporation‐mediated synthetic DNA delivery, thus enabling direct in vivo production of nanovaccines. DNA‐launched nanoparticles are demonstrated displaying an HIV immunogen spontaneously self‐assembled in vivo. DNA‐launched nanovaccines induce stronger humoral responses than their monomeric counterparts in both mice and guinea pigs, and uniquely elicit CD8+ effector T‐cell immunity as compared to recombinant protein nanovaccines. Improvements in vaccine responses recapitulate when DNA‐launched nanovaccines with alternative scaffolds and decorated antigen are designed and evaluated. Finally, evaluation of functional immune responses induced by DLnanovaccines demonstrates that, in comparison to control mice or mice immunized with DNA‐encoded hemagglutinin monomer, mice immunized with a DNA‐launched hemagglutinin nanoparticle vaccine fully survive a lethal influenza challenge, and have substantially lower viral load, weight loss, and influenza‐induced lung pathology. Additional study of these next‐generation in vivo‐produced nanovaccines may offer advantages for immunization against multiple disease targets.

Abstract Cyclodextrin glucanotransferases (CGTases) synthesize cyclic oligosaccharides (cyclodextrins, CD) from starch. A CGTase from Bacillus sp. G‐825‐6 was engineered by site‐directed mutagenesis at two positions by the construction of the variants Y183W, Y183R, D358R, Y183W/D358R, and Y183R/D358R. Among CD composed of 7–12 glucose units (CD7–CD12), Y183W mainly produced CD8. Y183R had completely lost its ability to synthesize CD7, and CD8 and the larger CD were the only cyclic oligosaccharides produced. D358R also formed mainly CD8–CD12 during a reaction time of 24 hr. The double mutant Y183W/D358R showed combined characteristics of the single mutations with very low CD7 cyclization activity and an increased formation of the larger CD. The results show that CGTases synthesizing mainly CD8–CD12 can be constructed allowing a convenient production of larger CD in significant amounts as host molecules in supramolecular complexing reactions.

Cytochrome P450s (CYPs) are heme-thiolated enzymes that catalyze the oxidation of C-H bonds in a regio- and stereo-selective manner. CYPs are widely present in the biological world. With the completion of more biological genome sequencing, the number and types of P450 enzymes have increased rapidly. P450 in microorganisms is easy to clone and express, rich in catalytic types, and strong in substrate adaptability, which has good application potential. Although the number of P450 enzymes found in microorganisms is huge, the function of most of the microorganism P450s has not been studied, and it contains a large number of excellent biocatalysts to be developed. This review is based on the P450 groups in microorganisms. First, it reviews the distribution of P450 groups in different microbial species, and then studies the application of microbial P450 enzymes in the pharmaceutical industry, chemical industry and environmental pollutant treatment in recent years. And focused on the application fields of P450 enzymes of different families to guide the selection of suitable P450s from the huge P450 library. In view of the current shortcomings of microbial P450 in the application process, the final solution is the most likely to assist the application of P450 enzymes in large-scale, that is, whole cell transformation combined with engineering, fusion P450 combined with immobilization technology., (© 2023 Wiley Periodicals LLC.)

Protocols for the construction of large, deeply mutagenized protein encoding libraries via Golden Gate assembly of synthetic DNA cassettes employ disparate, system-specific methodology. Here we present a standardized Golden Gate method for building user-defined libraries. We demonstrate that a 25 μL reaction, using 40 fmol of input DNA, can generate a library on the order of 1 × 10 6 members and that reaction volume or input DNA concentration can be scaled up with no losses in transformation efficiency. Such libraries can be constructed from dsDNA cassettes generated either by degenerate oligonucleotides or oligo pools. We demonstrate its real-world effectiveness by building custom, user-defined libraries on the order of 10 4 -10 7 unique protein encoding variants for two orthogonal protein engineering systems. We include a detailed protocol and provide several general-use destination vectors., (© 2023 Wiley Periodicals LLC.)

Structural information can help engineer enzymes. Usually, specific amino acids in particular regions are targeted for functional reconstruction to enhance the catalytic performance, including activity, stereoselectivity, and thermostability. Appropriate selection of target sites is the key to structure-based design, which requires elucidation of the structure-function relationships. Here, we summarize the mutations of residues in different specific regions, including active center, access tunnels, and flexible loops, on fine-tuning the catalytic performance of enzymes, and discuss the effects of altering the local structural environment on the functions. In addition, we keep up with the recent progress of structure-based approaches for enzyme engineering, aiming to provide some guidance on how to take advantage of the structural information., (© 2023 Wiley Periodicals LLC.)

D-Amino acid oxidase (DAAO) selectively catalyzes the oxidative deamination of  D-amino acids, making it one of the most promising routes for synthesizing optically pure  L-amino acids, including  L-phosphinothricin ( L-PPT), a chiral herbicide with significant market potential. However, the native DAAOs that have been reported have low activity against unnatural acid substrate  D-PPT. Herein, we designed and screened a DAAO from Rhodotorula taiwanensis (RtwDAAO), and improved its catalytic potential toward  D-PPT through protein engineering. A semirational design approach was employed to create a mutation library based on the tunnel-pocket engineering. After three rounds of iterative saturation mutagenesis, the optimal variant M 3rd -SHVG was obtained, exhibiting a >2000-fold increase in relative activity. The kinetic parameters showed that M 3rd -SHVG improved the substrate binding affinity and turnover number. This is the optimal parameter reported so far. Further, molecular dynamics simulation revealed that the M 3rd -SHVG reshapes the tunnel-pocket and corrects the direction of enzyme-substrate binding, allowing efficiently catalyze unnatural substrates. Our strategy demonstrates that the redesign of tunnel-pockets is effective in improving the activity and kinetic efficiency of DAAO, which provides a valuable reference for enzymatic catalysis. With the M 3rd -SHVG as biocatalyst, 500 mM D, L-PPT was completely converted and the yield reached 98%. The results laid the foundation for further industrial production., (© 2023 Wiley Periodicals LLC.)