Chlorogenic acid (CGA) is a valuable phenolic acid with various pharmaceutical functions. In our previous study, de novo synthesis of CGA in Saccharomyces cerevisiae was achieved. However, its yield required improvement before large scale production. In this study, systematic metabolic engineering strategy was used to reconstruct chassis cell S. cerevisiae YC0707 to enhance its CGA yield from glucose. To balance the supply of phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P), ZWF1 (encoding glucose-6-phosphate dehydrogenase) and GND1 (encoding 6-phosphogluconate dehydrogenase) were overexpressed by strong promoter P TEF1 swapping, thereby strengthening the pentose phosphate pathway. The mutant of phosphofructokinase (PFK2 S718D ) was further introduced to weaken the glycolytic pathway. Then, the p-coumaric acid synthesis capacity was enhanced by employing tyrosine ammonia lyase from Rhodotorula glutinis (RgTAL), ΔHAM1, and ΔYJL028W. Fusion expression of AtC4H (cinnamate-4-hydroxylase) and At4CL1 (4-coumarate CoA ligase 1), together with CsHQT (hydroxycinnamoyl CoA quinate transferase) and AtC3'H (p-coumaroyl shikimate 3-hydroxylase), improved biosynthetic flux to CGA. Subsequently, the microenvironment of P450 enzymes was improved by overexpressing INO2 (a transcription factor for lipid biosynthesis) and removal of heme oxygenase gene HMX1. Furthermore, screening potential transporters to facilitate CGA accumulation. Finally, we optimized the fermentation conditions. Using these strategies, CGA titers increased from 234.8 mg/L to 837.2 mg/L in shake flasks and reached 1.62 g/L in a 5-L bioreactor, representing the highest report in S. cerevisiae and providing new insights for CGA production.

As antimicrobial resistance increases, urinary tract infections (UTIs) are expected to pose an increased burden in morbidity and expense on the health care system, increasing the need for alternative antibiotic-sparing treatments. Most UTIs are caused by uropathogenic Escherichia coli (UPEC), whereas Klebsiella pneumoniae causes a large portion of non-UPEC UTIs. Both bacteria express type 1 pili tipped with the mannose-binding FimH adhesin critical for UTI pathogenesis. We generated and biochemically characterized 33 murine monoclonal antibodies (mAbs) to FimH. Three mAbs protected mice from E. coli UTI. Mechanistically, we show that this protection is Fc independent and mediated by the ability of these mAbs to sterically block FimH function by recognizing a high-affinity FimH conformation. Our data reveal that FimH mAbs hold promise as an antibiotic-sparing treatment strategy.

IDH1 mutation occurs early in glioma development; thus, mutant IDH1-specific inhibitors are being developed as glioma therapy. But, recent reports suggest that mutant IDH1 inhibitors treatments result in loss of therapeutic vulnerabilities and makes cells resistant to anticancer agents. To overcome resistance, the new paradigm in drug discovery is to develop molecules that can degrade oncogenes by harnessing cellular ubiquitination machinery. Therefore, it is imperative to identify strategies for degrading mutant IDH1 employing cellular ubiquitination machinery. To address this, we found that concerted action of the mitotic kinases Cdk1/Cyclin B1 and Plk1 increases mutant IDH1 enzyme activity. It is known that phosphorylation is linked to protein stability, phosphorylation of a short linear degron motif or phosphodegron can trigger target protein ubiquitination. By contrast, phosphorylation of constitutively active degron motifs can block target protein ubiquitination - phospho-inactivated degron. As phosphorylation can trigger or block ubiquitination, it is unknown if phosphorylation affects mutant IDH1 ubiquitination in mitosis. Therefore, in this study, we asked if phosphorylation of mutant IDH1 is linked to ubiquitination. To answer this question, we examined ubiquitination of phosphomutants and phosphomimetics of mutant IDH1 in mitosis. We found that blocking IDH1R132H phosphorylation is linked to ubiquitination. We observed that APC/C CDH1 ubiquitinates IDH1R132H-T77A-S94A. Further, we show that APC/C CDH1 ubiquitinates lysines 301 and 321 in C-terminal domain of IDH1R132H-T77A-S94A. Thus, blocking mutant IDH1 phosphorylation triggers APC/C CDH1-dependent ubiquitination in mitotic cells. We suggest employing mitotic inhibitors that also block phosphorylation of mutant IDH1 can ubiquitinate mutant IDH1 in cancer cells.

Industrial biotechnology offers a potential ecological solution for PET recycling under relatively mild reaction conditions via enzymatic degradation, particularly using the leaf branch compost cutinase (LCC) quadruple mutant ICCG. To improve the efficient downstream processing of this biocatalyst after heterologous gene expression with a suitable production host, protein crystallization can serve as an effective purification/capture step. Enhancing protein crystallization was achieved in recent studies by introducing electrostatic (and aromatic) interactions in two homologous alcohol dehydrogenases (Lb/LkADH) and an ene reductase (NspER1-L1,5) produced with Escherichia coli. In this study, ICCG, which is difficult to crystallize, was utilized for the application of crystal contact engineering strategies, resulting in ICCG mutant L50Y (ICCGY). Previously focused on the Lys-Glu interaction for the introduction of electrostatic interactions at crystal contacts, the applicability of the engineering strategy was extended here to an Arg-Glu interaction to increase crystallizability, as shown for ICCGY T110E. Furthermore, the rationale of the engineering approach is demonstrated by introducing Lys and Glu at non-crystal contacts or sites without potential interaction partners as negative controls. These resulting mutants crystallized comparably but not superior to the wild-type protein. As demonstrated by this study, crystal contact engineering emerges as a promising approach for rationally enhancing protein crystallization. This advancement could significantly streamline biotechnological downstream processing, offering a more efficient pathway for research and industry.

Heterotrimeric Gα:Gβγ G proteins function as molecular switches downstream of G protein-coupled receptors (GPCRs). They alternate between a heterotrimeric GDP-bound OFF-state and a GTP-bound ON-state in which GαGTP is separated from the Gβγ dimer. Consequently, pharmacological tools to securely prevent the OFF-ON transition are of utmost importance to investigate their molecular switch function, specific contribution to GPCR signal transduction, and potential as drug targets. FR900359 (FR) and YM-254890 (YM), two natural cyclic peptides and highly specific inhibitors of Gq/11 heterotrimers, are exactly such tools. To date, their efficient and long-lasting inhibition of Gq/11 signaling has been attributed solely to a wedge-like binding to Gα, thereby preventing separation of the GTPase and α-helical domains and thus GDP release. Here, we use X-ray crystallography, biochemical and signaling assays, and BRET-based biosensors to show that FR and YM also function as stabilizers of the Gα:Gβγ subunit interface. Our high-resolution structures reveal a network of residues in Gα and two highly conserved amino acids in Gβ that are targeted by FR and YM to glue the Gβγ complex to the inactive GαGDP subunit. Unlike all previously developed nucleotide-state specific inhibitors that sequester Gα in its OFF-state but compete with Gβγ, FR and YM actively promote the inhibitory occlusion of GαGDP by Gβγ. In doing so, they securely lock the entire heterotrimer, not just Gα, in its inactive state. Our results identify FR and YM as molecular glues for Gα and Gβγ that combine simultaneous binding to both subunits with inhibition of G protein signaling.

The enzyme 2-deoxy-D-ribose-5-phosphate aldolase (DERA) naturally catalyzes the reversible aldol addition between acetaldehyde and D-glyceraldehyde-3-phosphate to yield 2-deoxy-D-ribose-5-phosphate. Herein we describe the redesign of DERA into a proficient non-natural peroxygenase that promotes the asymmetric epoxidation of various α,β-unsaturated aldehydes. This repurposed aldolase, named DERA-EP, is able to utilize H2O2 to accomplish both anti- and syn-selective epoxidations of various α,β-unsaturated aldehydes to give the corresponding epoxides with moderate to high diastereoselectivity (diastereomeric ratio up to 99 : 1) and excellent enantioselectivity (enantiomeric ratio up to 99 : 1). Crystallographic analysis of DERA-EP in a substrate-free and substrate-bound state provides a structural context for the evolved activity, a clear explanation for the high enantioselectivity, and compelling evidence for catalysis via enzyme-bound iminium ion intermediates. The unprecedented anti-selectivity of DERA-EP with multiple α,β-unsaturated aldehydes is complementary to the syn-selectivity of previously reported enzyme-, metal- and organo-catalysts, making DERA-EP an attractive new asset to the toolbox of epoxidation catalysts.

Klebsiella pneumoniae carbapenemases (KPCs) have spread and diversified extensively. To date, 242 clinical variants have been identified and harbor different hydrolytic capacities, thereby interfering with rapid diagnostic tests. The accurate detection of KPC variants is crucial to guide treatment and control measures in healthcare settings. We constructed KPC variants to assess the mutational impact on detection capacities of resistance-based tests. KPC variants (n = 45) were characterized phenotypically and used to measure the detection sensitivity of KPC detection methods (two lateral flow immunoassays [LFIAs], three hydrolysis tests, three selective culture media, and two PCR-based tests). We identified four antibiotic susceptibility patterns: "KPC-like" (23/45; 51%), "extended-spectrum beta-lactamase-like" (6/45; 13%), "ceftazidimase" (9/45; 20%), and outlier variants with "mixed-profiles" (5/45; 11%). These phenotypes had different impacts on the detection capabilities of hydrolysis tests (0%-100%), LFIA (44%-100%), and selective culture media (0%-100%), highlighting a risk of misdiagnosis for some KPC variants. All variants were detected with PCR-based tests. To detect the maximum of KPC variants, fecal carriage screening requires a combination of selective media targeting resistance to carbapenems, third-generation cephalosporins, and ceftazidime-avibactam. From antibiotic susceptibility testing, resistance to ceftazidime ± avibactam and specific phenotypic profiles should be used as warnings to track the presence of KPC variants. We recommend LFIA as a first-line test, owing to its high sensitivity in detecting KPC variants. Nevertheless, using a combination of tests may remain wise in some situations. The spread of KPC variants remains a significant concern, particularly as reversion to ancestral phenotype could restore carbapenem resistance and lead to therapeutic failure.

Drought is one of the major environmental stresses limiting crop growth and yield. Epigenetic regulations play crucial roles in plant adaptation to environmental changes, whereas the epigenetic mechanism of drought resistance in crops remains largely elusive. Here, we report that the nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase TaSRT1 negatively regulates drought tolerance in wheat. Compared with the wild type, the tasrt1 mutant had higher relative water contents, along with a smaller stomatal aperture and improved water use efficiency under drought conditions, whereas TaSRT1 overexpression plants exhibited opposite phenotypes. TaSRT1 directly interacted with the drought-resistant pivotal factor TaDT-A to regulate its protein stability and transcriptional activity through lysine deacetylation. Furthermore, the key lysine residue of TaDT-A was identified as a deacetylation/acetylation site that plays an important role in regulating its stability. In addition, genetic analysis indicated TaDT-A functions downstream of TaSRT1 to modulate drought resistance. These findings uncover how the functional interplay between epigenetic regulator and transcription factors regulates drought resistance in plants, and illustrate a mechanism by which lysine deacetylase affects gene transcription via influencing non-histone protein acetylation and regulating their function.

Isobutene is a promising precursor for jet fuel due to its high energy density and favorable combustion properties. Light-driven bioproduction of isobutene has recently been investigated as an alternative strategy to crude oil refinement or fermentation-based manufacturing processes by harnessing the unicellular cyanobacterium Synechocystis sp. PCC 6803 and the α-ketoisocaproate dioxygenase (RnKICD) from Rattus norvegicus. However, the obtained production level was not sufficient, partially due to the promiscuous activity of RnKICD. The enzyme catalyzes both the reaction with ρ-hydroxyphenylpyruvate (HPP) for homogentisate formation, as well as the reaction with α-ketoisocaproate (KIC), the precursor for isobutene synthesis. Here, to overcome this bottleneck step in the isobutene biosynthesis, protein engineering was employed to improve RnKICD activity and in vivo isobutene production. Purified RnKICD variants were characterized by measuring in vitro KIC and HPP consumption rates, as well as isobutene formation rate. The active site mutations F336V, N363A altered the KIC and HPP consumption rates, while the KIC-to-isobutene conversion ratio was only marginally affected. Besides, the RnKICD variants F336V, N363A and F336V/N363A exhibited a substantially enhanced substrate selectivity for KIC over HPP. Among the examined engineered Synechocystis strains, Syn-F336V showed a 4-fold improvement in isobutene production, compared to the base strain (Syn-RnKICD). Our findings reveal that residues F336 and N363 play a crucial role in substrate interactions, as targeted mutations at these sites shifted the substrate selectivity towards KIC while F336V elevated the in vivo isobutene production levels significantly. We conclude that engineering the active site of RnKICD is a potent tool for improving isobutene bioproduction in Synechocystis.

Multidrug resistance proteins (MRPs) are one of the major mechanisms for developing cancer drug resistance. Human MRP4 (hMRP4) plays an important role in various chemotherapy-resistant cancers. Here, we show hMRP4 mediates the resistance of a broad spectrum of antitumor reagents in the cultured tumor cells, among which the cell resistance to vincristine and 5-fluorouracil is rescued by supplementing a tyrosinase inhibitor, lapatinib. The cryoelectron microscopy (cryo-EM) structures of hMRP4 in the substrate- or inhibitor-bound form are determined. Although lapatinib shares partial binding sites with vincristine and 5-fluorouracil using a similar set of crucial residues located in the central cavity of hMRP4, the high binding affinity of lapatinib and its unique binding mode with transmembrane helices TM2 and TM12 inside the pathway tunnel prohibit hMRP4 from structural transition between intermediate states during drug translocation. This study provides mechanistic insights into the therapeutical potential of lapatinib in combating hMRP4-mediated MDR.

In vivo genetic diversifiers have previously enabled efficient searches of genetic variant fitness landscapes for continuous directed evolution. However, existing genomic diversification modalities for mammalian genomic loci exclusively rely on deaminases to generate transition mutations within target loci, forfeiting access to most missense mutations. Here, we engineer CRISPR-guided error-prone DNA polymerases (EvolvR) to diversify all four nucleotides within genomic loci in mammalian cells. We demonstrate that EvolvR generates both transition and transversion mutations throughout a mutation window of at least 40 bp and implement EvolvR to evolve previously unreported drug-resistant MAP2K1 variants via substitutions not achievable with deaminases. Moreover, we discover that the nickase's mismatch tolerance limits EvolvR's mutation window and substitution biases in a gRNA-specific fashion. To compensate for gRNA-to-gRNA variability in mutagenesis, we maximize the number of gRNA target sequences by incorporating a PAM-flexible nickase into EvolvR. Finally, we find a strong correlation between predicted free energy changes underlying R-loop formation and EvolvR's performance using a given gRNA. The EvolvR system diversifies all four nucleotides to enable the evolution of mammalian cells, while nuclease and gRNA-specific properties underlying nickase fidelity can be engineered to further enhance EvolvR's mutation rates.

Adenosine triphosphate (ATP)-dependent in vitro bioprocesses, such as cell-free protein synthesis and the production of phosphorylated fine chemicals, are of considerable industrial significance. However, their implementation is mainly hindered by the high cost of ATP. We propose and demonstrate the feasibility of a cell-free ATP regeneration system based on the in situ generation of the high-energy compound acetyl phosphate from low-cost d-fructose and inorganic phosphate substrates. The enzyme cascade chains d-fructose phosphoketolase, d-erythrose isomerase, d-erythrulose phosphoketolase, and glycolaldehyde phosphoketolase activities theoretically enabling production of 3 mol ATP per mol of d-fructose. Through a semirational engineering approach and the screening of nine single-mutation libraries, we optimized the phosphoketolase (PKT) from Bifidobacterium adolescentis, identifying the improved variant Bad.F6Pkt H548N. This mutant exhibited a 5.6-fold increase in d-fructose activity, a 2.2-fold increase in d-erythrulose activity, and a 1.3-fold increase in glycolaldehyde activity compared to the wild-type enzyme. The Bad.F6Pkt H548N mutant was initially implemented in a cell-free reaction system together with an acetate kinase from Geobacillus stearothermophilus and a glycerol kinase from Cellulomonas sp. for the production of glycerol-3 phosphate from ADP and glycerol. We demonstrated the feasibility of ATP regeneration from 25 mM d-fructose with a stoichiometry of 1 mol of ATP per mol of C6 ketose. Subsequently, the reaction system was enhanced by incorporating d-erythrose isomerase activity provided by a l-rhamnose isomerase from Pseudomonas stutzeri. In the complete system, the ATP yield increased to 2.53 mol molfructose-1 with a maximum productivity of 7.2 mM h-1.

Bioremediation relying on highly efficient degrading bacteria constitutes a promising and sustainable avenue for controlling and reducing nitrophenol contamination in the environment. A thorough understanding of the bacterial degradation mechanism of nitrophenol is of paramount importance for supporting the development of efficient microbial remediation technology.

In this study, a new bacterium, Rhodococcus sp. 21391, endowed with superior p-nitrophenol (PNP) degradation ability was obtained. Genomic and comparative proteomic analyses revealed that it utilizes the 1,2,4-benzenetriol (BT) pathway for PNP degradation. The catalytic properties of the two-component p-nitrophenol monooxygenase RsNcpAB from the strain were investigated in vitro. The enzyme exhibited a broad substrate selectivity, catalyzing the oxidation of various nitrophenols and halogenated phenols, with significant potential for further research and development. Additionally, the crystal structure of the oxidative component of p-nitrophenol monooxygenase, RsNcpA, was determined. Structural analysis and site-directed mutagenesis revealed that residues Arg100 and His293 in the active site play a crucial role in enzyme catalysis, and a catalytic mechanism model was subsequently proposed.

This study reports a high-performance nitrophenol-degrading bacterium and enzyme, and reveals their mechanisms at the molecular level. These findings increase the understanding of the bacterial degradation of nitrophenol, thereby providing a crucial foundation for the development of efficient bioremediation technologies.

Uricase is a key enzyme in purine metabolism that catalyzes the oxidation of uric acid to allantoin, widely used in the treatment of hyperuricemia and gout. In this study, error-prone PCR and one high-throughput screening method were employed to generate uricase mutants with enhanced enzymatic activity from Aspergillus flavus and Candida utilis. After several rounds of mutation and selection, an A. flavus uricase mutant, af-UAM, with activity of 46.21 U/mg, and a C. utilis uricase mutant, cu-UAM, with activity of 31.43 U/mg, were obtained-representing the highest uricase activities reported up to date. Site-directed mutagenesis revealed that the Thr231Ala substitution in A. flavus uricase and the Val234Met substitution in C. utilis uricase were key factors driving their enhanced activities. Furthermore, in vivo experiments demonstrated significant clinical potential of these mutants. These findings offer new insights into the structure-function relationship of uricase and present promising candidates for therapeutic applications in hyperuricemia treatment.

Salidroside is a phenylpropanoid glycoside with wide applications in the food, pharmaceutical, and cosmetic industries; however, the plant genus Rhodiola, the natural source of salidroside, has slow growth and limited distribution. In this study, we designed a novel six-enzyme biocatalytic cascade for the efficient production of salidroside, utilizing cost-effective bio-based L-Tyrosine as the starting material. A preliminary analysis revealed that the poor thermostability of the Bacillus licheniformis UDP-glycosyltransferase (EC 2.4.1.384) BlYjiC M6 is a bottleneck in the cascade. Therefore, a combined computational strategy was used to engineer it and finally obtained a mutant TSM6 (T304V/G307A/N309W/F123W/T344V/D271G) with a 134-fold longer half-life at 40 °C and a 13 °C higher Tmapp compared to M6. The integration of TSM6 into the cascade improved salidroside productivity significantly, while reducing residual intermediates. After further optimization, the whole-cell biocatalytic cascade achieved a high salidroside titer of 12.8 g·L-1 in a 5 L bioreactor, giving a productivity of 0.53 g·L-1·h-1. This study provides a green and efficient biosynthetic process for salidroside production and highlights the potential of enzyme engineering to enhance the biocatalytic cascade.

Epoxides, a class of ethers with a three-membered ring structure, are widely used in the textile, pharmaceutical, and packaging industries. Chemoenzymatic epoxidation presents a promising method for synthesizing epoxides. However, its epoxidation efficiency is hindered by low chemoselective perhydrolysis, which is caused by the hydrolysis side reaction in the aqueous phase. In this study, a chemoenzymatic epoxidation process in the aqueous phase was developed by utilizing an acyltransferase from P. litoralis (PlAcT) for its chemoselective perhydrolysis. Crystal structure analysis, molecular dynamics simulations, and quantum mechanics calculations, along with site-specific mutagenesis, revealed that the selectivity of perhydrolysis is due to a lower energy barrier in the acyl transfer step compared to that in hydrolysis. Furthermore, the mutant PlAcTM3-2 exhibited a 7.6-fold improvement in solvent stability and a 1.3-fold increase in perhydrolysis activity compared to the wild type, achieved by reshaping interface interactions. As a result, the engineered strain Y07, harboring PlAcTM3-2, successfully synthesized compounds 3a-3n with conversions ranging from 11-99%, and the titers of compounds α-pinene oxide(3i), β-pinene oxide(3j), 3-carene oxide(3k), and limonene dioxide(3l-3) reached 55.8, 16.7, 75.2, and 21.4 g/L, respectively. These results demonstrate a sustainable method for chemoenzymatic epoxidation in the aqueous phase.

tRNAIle2 lysidine synthetase (TilS) is a bacterial-specific wobble-modifying enzyme that acts on the isoleucine-accepting tRNAIle2 CAU. TilS installs a lysine at the C34 position of the anticodon, generating the lysidine modification. The resulting LAU anticodon enables exclusive decoding of infrequently used AUA isoleucine codons, rejecting AUG methionine codons. Compared to other wobble-modifying enzymes that contact only the anticodon arm of their cognate tRNAs, TilS is distinct in containing additional domains outside of the N-terminal active site. For type I TilS enzymes such as the B. cenocepacia TilS (BcTilS) investigated here, appended domains contact the tRNAIle2 substrate along the body and through the acceptor stem, up to 60 Å away from the target C34. Among bacterial tRNAs, only unmodified tRNAIle2 and tRNAMet share an anticodon, suggesting that the appended domains of TilS provide substrate recognition strategies that other wobble-modifying enzymes do not need. Here, we investigate both protein and tRNA elements to understand the strategy by which TilS accepts its cognate tRNAIle2 substrate and rejects the near-cognate tRNAMet.

Protein crystallization is an alternative to well-established but cost-intensive and time-consuming chromatography in biotechnological processes, with protein crystallization defined as an essential unit operation for isolating proteins, e.g., active pharmaceutical ingredients. Crystalline therapeutic proteins attract interest in formulation and delivery processes of biopharmaceuticals due to the high purity, concentration, and stability of the crystalline state. Although improving protein crystallization is mainly achieved by high-throughput screening of crystallization conditions, recent studies have established a rational protein engineering approach to enhance crystallization for two homologous alcohol dehydrogenases from Lactobacillus brevis (LbADH) and Lactobacillus kefiri (LkADH). As generalizing crystallization processes across a wide range of target proteins remains challenging, this study takes a further step by applying the successful crystal contact engineering strategies for LbADH/LkADH to a non-homologous protein, an NADH-binding derivative of the Nostoc sp. PCC 1720 ene reductase (NspER1-L1,5). Here, the focus lies on introducing electrostatic interactions at crystal contacts, specifically between lysine and glutamic acid. Out of the nine tested NspER1-L1,5 mutants produced in E. coli, six crystallized, while four mutants revealed an increased propensity to crystallize in static µL-batch crystallization compared to the wild type: Q204K, Q350K, D352K, and T354K. The best-performing mutant Q204K was selected for upscaling, crystallizing faster than the wild type in a stirred batch crystallizer. Even when spiked with E. coli cell lysate, the mutant maintained increased crystallizability compared to the wild type. The results of this study highlight the potential of crystal contact engineering as a reliable tool for improving protein crystallization as an alternative to chromatography, paving the way for more efficient biotechnological downstream processing.

Site-directed mutagenesis is a fundamental tool indispensable for protein and plasmid engineering. An important technological question is how to achieve the ideal efficiency of 100%. Based on complementary primer pairs, the QuickChange method has been widely used, but it requires significant improvements due to its low efficiency and frequent unwanted mutations. An alternative and innovative strategy is to utilize primer pairs with 3'-overhangs, but this approach has not been fully developed. As the first step toward reaching the efficiency of 100%, we have optimized this approach systematically (such as use of newly designed short primers, test of different Pfu DNA polymerases, and modification of PCR parameters) and evaluated the resulting method extensively with >100 mutations on 12 mammalian expression vectors, ranging from 7.0 to 13.4 kb in size and encoding ten epigenetic regulators linked to cancer and neurodevelopmental disorders. We have also tested the new method with two expression vectors for the SARS-CoV-2 spike protein. Compared to the QuickChange method, the success rate has increased substantially, with an average efficiency of ∼50%, with some at or close to 100%, and requiring much less time for engineering various mutations. Therefore, we have developed a new site-directed mutagenesis method for efficient and economical generation of various mutations. Notably, the method failed with a human KAT2B expression plasmid that possesses extremely GC-rich sequences. Thus, this study also sheds light on how to improve the method for developing ideal mutagenesis methods with the efficiency of ∼100% for a wide spectrum of plasmids.

An open reading frame from the actinobacterium Mycolicibacterium smegmatis annotated as a Prostaglandin H Synthase (PGHS) was expressed with an N-terminal (his)6 tag and purified to homogeneity. The enzyme has a monomeric molecular weight of 68.3 kD and exists as a dimer in the presence of nonionic detergent. The enzyme uses saturated and unsaturated fatty acids as substrates and catalyzes two reactions: the addition of molecular oxygen alpha to the carboxylate group to form the 2-hydroperoxy fatty acid, followed by reduction to the 2-hydroxy fatty acid. The initial reduction reaction does not require a source of electrons, but electrons must be provided from an appropriate donor such as epinephrine for the reduction reaction to go to completion. Minor reaction products one carbon atom shorter than the original fatty acid substrate are also observed; These most likely arise from the spontaneous decarboxylation of the 2-hydroperoxy fatty acid product to form an aldehyde. This dual function dioxygenase/peroxidase is unusual among the lipid dioxygenases and may represent a bacterial precursor to mammalian PGHS.

The assembly of the 26S proteasome, a multi-subunit complex for regulated protein turnover, proceeds via the formation of intermediates. Giardia lamblia does not encode proteasome regulatory subunit Rpn12 or proteasome complex subunit Sem1, two proteins crucial for assembling the proteasome lid. To understand how the interactions between the giardial proteasome lid subunits may have changed to compensate for their absence, we used yeast two-hybrid to generate a binary interactome map of Giardia's lid subunits. Most interactions within the Giardia lid are stronger than Saccharomyces cerevisiae lid, which may compensate for Rpn12 and Sem1 absence. A notable exception was the weaker interaction between the two non-ATPase lid subunits, GlRpn11 and GlRpn8, compared to the strong interaction between yeast orthologs Rpn11 and Rpn8. The Rpn11-Rpn8 dimer provides a platform for lid assembly. Their interaction involves the insertion of a methionine residue of Rpn11 into a hydrophobic pocket of Rpn8. Molecular modeling indicates that GlRpn8's pocket is wider, reconciling the experimental observation of its weak interaction with GlRpn11. This weaker interaction may have evolved to support proteasome-independent functions of GlRpn11, which localizes to multiple subcellular regions, including the mitosomes, where other proteasome subunits cannot be detected. Functional complementation in yeast shows that GlRpn11 can influence mitochondrial function and distribution. Together these observations show that GlRpn11 functions at the mitosome. Thus, this parasite's proteasome lid has a simpler subunit architecture than that of yeast with structural attributes to support dual functionalities for GlRpn11. Such parasite-specific proteasome features provide opportunities for controlling parasite transmission.

(L)-2,4-dihydroxybutyrate (DHB) is a versatile compound that can serve as a precursor for the synthesis of the methionine analog 2-hydroxy-4-(methylthio)butyrate and new advanced polymers. We previously implemented in Escherichia coli an artificial biosynthetic pathway for the aerobic production of DHB from glucose, which relies on the deamination of (L)-homoserine followed by the reduction of 2-oxo-4-hydroxybutyrate (OHB) and yields DHB by an enzyme-bearing NADH-dependent OHB reductase activity. Under aerobic conditions, using NADPH as a cofactor is more favorable for reduction processes. We report the construction of an NADPH-dependent OHB reductase and increased intracellular NADPH supply by metabolic engineering to improve DHB production. Key cofactor discriminating positions were identified in the previously engineered NADH-dependent OHB reductase (E. coli malate dehydrogenase I12V:R81A:M85Q:D86S:G179D) and tested by mutational scanning. The two point mutations D34G:I35R were found to increase the specificity for NADPH by more than three orders of magnitude. Using the new OHB reductase enzyme, replacing the homoserine transaminase with the improved variant Ec.AlaC A142P:Y275D and increasing the NADPH supply by overexpressing the pntAB gene encoding the membrane-bound transhydrogenase yielded a strain that produced DHB from glucose at a yield of 0.25 molDHB molGlucose -1 in shake-flask experiments, which corresponds to a 50% increase compared to previous producer strains. Upon 24 h of batch cultivation of the most advanced DHB producer strain constructed in this work, a volumetric productivity of 0.83 mmolDHB L-1 h-1 was reached.

Mycobacterium tuberculosis is well adapted to survive and persist in the infected host, escaping the host's immune response. Since polyamines such as spermine, which are synthesized by infected macrophages, are able to inhibit the growth of M. tuberculosis, the pathogen needs strategies to cope with these toxic metabolites. The actinomycete Streptomyces coelicolor, a close relative of M. tuberculosis, makes use of a gamma-glutamylation pathway to functionally neutralize spermine. We therefore considered whether a similar pathway would be functional in M. tuberculosis. In the current study, we demonstrated that M. tuberculosis growth was inhibited by the polyamine spermine. Using in vitro enzymatic assays we determined that GlnA3Mt (Rv1878) possesses genuine gamma-glutamylspermine synthetase catalytic activity. We further showed that purified His-Strep-GlnA3Mt, as well as native GlnA3Mt, prefer spermine as a substrate over putrescine, cadaverine, spermidine, or other monoamines and amino acids, suggesting that GlnA3Mt may play a specific role in the detoxification of the polyamine spermine. However, the deletion of the glnA3 gene in M. tuberculosis did not result in growth inhibition or enhanced sensitivity of M. tuberculosis in the presence of high spermine concentrations. Gene expression analysis of spermine-treated M. tuberculosis revealed no difference in the level of glnA3Mt expression relative to untreated cells, whereas a gene encoding a previously characterized efflux pump (Mmr; rv3065) was significantly upregulated. This suggests that bacterial survival under elevated spermine concentrations can not only be achieved by detoxification of spermine itself but also by mechanisms resulting in decreased spermine levels in the bacteria.

Upon Mycobacterium tuberculosis infection macrophages synthesize the polyamine spermine, which at elevated concentrations is toxic for M. tuberculosis. Based on our investigations of spermine resistance in the closely related actinomycete Streptomyces coelicolor, we hypothesized that the glutamylspermine synthetase GlnA3 may be responsible for the resistance of M. tuberculosis against toxic spermine. Here we show that GlnA3Mt can indeed covalently modify spermine via glutamylation. However, GlnA3Mt is probably not the only resistance mechanism since a glnA3 null mutant of M. tuberculosis can survive under spermine stress. Gene expression studies suggest that an efflux pump may participate in resistance. Thus a combination of GlnA3Mt and specific efflux pumps acting as putative spermine transporters may constitute an active spermine-detoxification system in M. tuberculosis.

Site-directed mutagenesis is an essential tool in molecular biology, protein engineering, plasmid engineering and synthetic biology. While the QuickChange method has been one of the most employed methods for site-directed mutagenesis, it is hindered by low efficiency and frequent introduction of unwanted mutations at the primer sites, raising the urgent need for new, more efficient, and reliable methods. Here, we present an optimized site-directed mutagenesis protocol that leverages partially complementary primer pairs with 3'-overhangs to improve mutagenesis efficiency and reduce error rates. Our method significantly enhances success rates, achieving an average efficiency of ∼50% with some instances approaching the ideal threshold of 100%, while also minimizing the time required for mutant generation. Typically, only 3 colonies need to be analyzed per mutagenesis reaction, and a skillful trainee can engineer 1 to 2 dozen mutant plasmids within a week. In addition, with an in-house protocol for preparing highly competent bacterial cells, we have further increased the reliability and cost-effectiveness of the method. Notably, such competent cells have been kept in a liquid nitrogen tank for >12 years with minimal loss of competency. Thus, this refined method offers a robust, efficient, and scalable solution for high-precision gene modification in vitro, with broad applications in protein and plasmid engineering. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: In vitro site-directed mutagenesis using an optimized primer design strategy Basic Protocol 2: Preparation of high-efficiency chemocompetent DH5α cells for transformation of mutagenized plasmid products Basic Protocol 3: Transformation of chemocompetent DH5α cells and obtaining bacterial colonies with correctly mutagenized plasmid products Alternate Protocol: Transformation if glycerol stocks are unavailable.

The Phosphoprotein (P protein) of the rabies virus has multiple roles in virus replication. A critical function is to act as a cofactor in genome replication and mRNA production through binding via its N-terminal region to the L protein, the essential enzyme for mRNA and genome synthesis/processing, and via its C-terminal domain (PCTD) to the N protein and viral RNA (N-RNA) ribonucleoprotein complex. The binding site of the PCTD on the N protein is a disordered loop that is expected to be phosphorylated at Ser389. This interface may provide novel targets for antiviral approaches. Following an alanine scan of the peptide we selected two single site mutations that showed improved affinity and combined these mutations with a phosphomimetic (S389E) to produce double and triple mutants in the context of linear and cyclic peptides of the disordered loop, with the goal of generating a competitive peptide against the N-RNA complex. To assess the binding properties of the peptides we characterized their thermodynamics identifying complex properties of improved enthalpy but with compensating entropy for mutants and cyclized peptides. Nevertheless, a triple mutant shows 3.5-fold stronger affinity for PCTD than the full-length S389E N protein. Structural characterization of the triple mutant suggests the improved affinity may be due to trapping a favoured β-strand structure for binding to the PCTD. This novel peptide may serve as a template for the future design of antivirals.

[FeFe]-hydrogenases catalyze the reversible two-electron reduction of two protons to molecular hydrogen. Although these enzymes are among the most efficient H2-converting biocatalysts in nature, their catalytic cofactor (termed H-cluster) is irreversibly destroyed upon contact with dioxygen. The [FeFe]-hydrogenase CbA5H from Clostridium beijerinckii has a unique mechanism to protect the H-cluster from oxygen-induced degradation. The protective strategy of CbA5H was proposed based on a partial protein structure of CbA5H's oxygen-shielded form. Here, we present a cryo-EM structure of 2.2 Å resolution from the entire enzyme in its dimeric and active state and elucidate the structural parameters of the reversible cofactor protection mechanism. We found that both subunits of the homodimeric structure of CbA5H have a Zn2+-binding four-helix domain, which does not play a role in electron transport as described for other complex protein structures. Biochemical data instead confirm that two [4Fe-4S] clusters are responsible for electron transfer in CbA5H, while the identified zinc atom is critical for oligomerization and protein stability.

Enzymatic fuel cells (EFCs) are emerging as promising technologies in renewable energy and biomedical applications, utilizing enzyme catalysts to convert the chemical energy of renewable biomass into electrical energy, known for their high energy conversion efficiency and excellent biocompatibility. Currently, EFCs face challenges of poor stability and catalytic efficiency at the cathodes, necessitating solutions to enhance the oriented immobilization of multicopper oxidases for improved heterogeneous electron transfer efficiency. This study successfully identified a surface-binding peptide (SBP, 13 amino acids) derived from a methionine-rich fragment (MetRich, 53 amino acids) in E. coli CueO through semirational design. The first phase of engineering focused on the structural characteristics of MetRich, pinpointing fragment N394-H406 (SBP 1.0, corresponding to variant CueO-M12) as the key region dominating the binding. Subsequent site-saturation mutagenesis, combined with electrochemical screening, yielded three variants, and among them, the variant CueO-M12-1 (CueO-M12 H398I) exhibited a more uniform favorable orientation with a 1.38-fold increase in current density. Further electrocatalytic kinetics analysis revealed a significant 21.2-fold improvement in kinetics current density (Jk) compared with that of CueO-WT, leading to the development of SBP 2.0. When SBPs were fused to laccase from Bacillus pumilus (BpL) and fungal bilirubin oxidase from Myrothecium verrucaria (MvBOD), respectively, they transformed a sluggish adsorption process into a rapid and oriented one. In addition, compared with SBP 1.0, SBP 2.0 endows BpL and MvBOD with enhanced electrocatalytic capabilities for oxygen reduction and glucose/O2 EFC performance. The engineered SBPs are promising for serving as a versatile "glue" to enable the immobilization of oxidoreductases in an oriented manner, which leads to a breakthrough in bioelectrocatalysis and thereby overcoming the current bottleneck of EFCs.

Esters are valuable aroma compounds and can be produced enzymatically by Baeyer-Villiger monooxygenases (BVMOs) from (aliphatic) ketone precursors. However, a genetically encoded biosensor system for the assessment of BVMO activity and the detection of reaction products is missing. In this work, we assembled a synthetic enzyme cascade - featuring an esterase, an alcohol dehydrogenase, and LuxAB - in the heterologous host Escherichia coli. Target esters are produced by a BVMO, subsequently cleaved, and the corresponding alcohol oxidized through the artificial pathway. Ultimately, aldehyde products are detected in vivo by LuxAB, a luciferase from Photorhabdus luminescens that emits bioluminescence upon the oxidation of aldehydes to the corresponding carboxylates. This biosensor system greatly accelerated the screening and selection of active BVMO variants from a focused library, omitting commonly used low-throughput chromatographic analysis. Engineered enzymes accepted linear aliphatic ketones such as 2-undecanone and 2-dodecanone and exhibited improved ester formation.

Volume-regulated anion channels (VRACs) are multimeric proteins composed of different paralogs of the LRRC8 family. They are activated in response to hypotonic swelling, but little is known about their specific functions. We studied two human individuals with the same congenital syndrome affecting blood vessels, brain, eyes, and bones. The LRRC8C gene harbored de novo variants in both patients, located in a region of the gene encoding the boundary between the pore and a cytoplasmic domain, which is depleted of sequence variations in control subjects. When studied by cryo-EM, both LRRC8C mutant proteins assembled as their wild-type counterparts, but showed increased flexibility, suggesting a destabilization of subunit interactions. When co-expressed with the obligatory LRRC8A subunit, the mutants exhibited enhanced activation, resulting in channel activity even at isotonic conditions in which wild-type channels are closed. We conclude that structural perturbations of LRRC8C impair channel gating and constitute the mechanistic basis of the dominant gain-of-function effect of these pathogenic variants. The pleiotropic phenotype of this novel clinical entity associated with monoallelic LRRC8C variants indicates the fundamental roles of VRACs in different tissues and organs.

Designing proteins with improved functions requires a deep understanding of how sequence and function are related, a vast space that is hard to explore. The ability to efficiently compress this space by identifying functionally important features is extremely valuable. Here we establish a method called EvoScan to comprehensively segment and scan the high-fitness sequence space to obtain anchor points that capture its essential features, especially in high dimensions. Our approach is compatible with any biomolecular function that can be coupled to a transcriptional output. We then develop deep learning and large language models to accurately reconstruct the space from these anchors, allowing computational prediction of novel, highly fit sequences without prior homology-derived or structural information. We apply this hybrid experimental-computational method, which we call EvoAI, to a repressor protein and find that only 82 anchors are sufficient to compress the high-fitness sequence space with a compression ratio of 1048. The extreme compressibility of the space informs both applied biomolecular design and understanding of natural evolution.

Cellular target engagement technologies enable quantification of intracellular drug binding; however, simultaneous assessment of drug-associated phenotypes has proven challenging. Here, we present cellular target engagement by accumulation of mutant as a platform that can concomitantly evaluate drug-target interactions and phenotypic responses using conditionally stabilized drug biosensors. We observe that drug-responsive proteotypes are prevalent among reported mutants of known drug targets. Compatible mutants appear to follow structural and biophysical logic that permits intra-protein and paralogous expansion of the biosensor pool. We then apply our method to uncouple target engagement from divergent cellular activities of MutT homolog 1 (MTH1) inhibitors, dissect Nudix hydrolase 15 (NUDT15)-associated thiopurine metabolism with the R139C pharmacogenetic variant, and profile the dynamics of poly(ADP-ribose) polymerase 1/2 (PARP1/2) binding and DNA trapping by PARP inhibitors (PARPi). Further, PARP1-derived biosensors facilitated high-throughput screening for PARP1 binders, as well as multimodal ex vivo analysis and non-invasive tracking of PARPi binding in live animals. This approach can facilitate holistic assessment of drug-target engagement by bridging drug binding events and their biological consequences.

The coordination of cell cycle progression and flagellar synthesis is a complex process in motile bacteria. In γ-proteobacteria, the localization of the flagellum to the cell pole is mediated by the SRP-type GTPase FlhF. However, the mechanism of action of FlhF, and its relationship with the cell pole landmark protein HubP remain unclear. In this study, we discovered a novel protein called FipA that is required for normal FlhF activity and function in polar flagellar synthesis. We demonstrated that membrane-localized FipA interacts with FlhF and is required for normal flagellar synthesis in Vibrio parahaemolyticus, Pseudomonas putida, and Shewanella putrefaciens, and it does so independently of the polar localization mediated by HubP. FipA exhibits a dynamic localization pattern and is present at the designated pole before flagellar synthesis begins, suggesting its role in licensing flagellar formation. This discovery provides insight into a new pathway for regulating flagellum synthesis and coordinating cellular organization in bacteria that rely on polar flagellation and FlhF-dependent localization.

Tylosin is an important macrolide antibiotic produced by Streptomyces fradiae. In the biosynthesis of tylosin, macrocin O-methyltransferase TylF catalyzes the conversion of the side-product tylosin C (macrocin) to the primary component tylosin A (C/A conversion). This conversion is the rate-limiting step in the biosynthesis of tylosin, and affects the quality of the end product. To find a high activity and environment-adapted TylF enzyme, a TylF variant pool has been constructed via protein evolution approach in our previous study (Fan et al., 2023 [41]). In this study, the TylF variants with higher C/A conversion rates were expressed in E. coli and purified. The variants TylFY139F, TylFQ138H, F232Y and TylFT36S, V54A were shown to have a higher C/A conversion rate at 30 °C than that of TylF at 38 °C. Moreover, they had a greater acid resistance and showed more adaptable to the pH change during fermentation. Further protein structural and substrate-binding affinity analyses revealed that the T36S, V54A, Q138H, Y139F, and F232Y mutations enlarged the volume of the substrate-binding pocket, thereby increasing the affinity of enzyme variants for their substrates of SAM and macrocin, and decreasing the inhibition of SAH. Three of the TylF variants were overexpressed in the industrial tylosin-producing S. fradiae strain, and the recombinant strains showed the highest C/A conversion at 30 °C without heating up to 38 °C during the last 24 h of fermentation. This is of great energy-saving significance for tylosin industrial production.