α-Glucosidase Inhibitory Activities of Lutein and Zeaxanthin Purified from Green Alga Chlorella ellipsoidea

α-Glucosidase inhibitors are used therapeutically to treat type-2 diabetes mellitus. Through a bioassay-guided fractionation technique, three carotenoids,(all-E)-lutein,(all-E)-zeaxanthin and(9-Z)-zeaxanthin, were purified from the green alga Chlorella ellipsoidea, in which(all-E)-lutein and(9-Z)-zeaxanthin had potent α-glucosidase inhibitory activity. IC_(50) values of(all-E)-lutein and(9-Z)-zeaxanthin were 70 and 53.5 μmol L^(-1) against Saccharomyces cerevisiae α-glucosidase, respectively, with non-competitive inhibition. In addition, IC_(50) values of(9-Z)-zeaxanthin against Bacillus stearothermophilus and rat-intestinal α-glucosidase were 805.1 and 671.2 μmol L^(-1), respectively. The K_i values of(all-E)-lutein and(9-Z)-zeaxanthin against S. cerevisiae α-glucosidase were 78.1 and 16.5 μmol L^(-1), respectively. Therefore, C. ellipsoidea carotenoids might be utilized as a novel candidate to prevent type-2 diabetes mellitus related disorders in food and medical industry.

A flexible,modular and versatile functional part assembly toolkit for gene cluster engineering in Streptomyces

Streptomyces has enormous potential to produce novel natural products(NPs)as it harbors a huge reservoir of uncharacterized and silent natural product biosynthetic gene clusters(BGCs).However,the lack of efficient gene cluster engineering strategies has hampered the pace of new drug discovery.Here,we developed an easy-to-use,highly flexible DNA assembly toolkit for gene cluster engineering.The DNA assembly toolkit is compatible with various DNA assembling approaches including Biobrick,Golden Gate,CATCH,yeast homologous recombination-based DNA assembly and homing endonuclease-mediated assembly.This compatibility offers great flexibility in handling multiple genetic parts or refactoring large gene clusters.To demonstrate the utility of this toolkit,we quantified a library of modular regulatory parts,and engineered a gene cluster(act)using characterized promoters that led to increased production.Overall,this work provides a powerful part assembly toolkit that can be used for natural product discovery and optimization in Streptomyces.

Functional predictability of universal gene circuits in diverse microbial hosts

Although the principles of synthetic biology were initially established in model bacteria,microbial producers,extremophiles and gut microbes have now emerged as valuable prokaryotic chassis for biological engineering.Extending the host range in which designed circuits can function reliably and predictably presents a major challenge for the concept of synthetic biology to materialize.In this work,we systematically characterized the cross-species universality of two transcriptional regulatory modules—the T7 RNA polymerase activator module and the repressors module—in three non-model microbes.We found striking linear relationships in circuit activities among different organisms for both modules.Parametrized model fitting revealed host non-specific parameters defining the universality of both modules.Lastly,a genetic NOT gate and a band-pass filter circuit were constructed from these modules and tested in non-model organisms.Combined models employing host non-specific parameters were successful in quantitatively predicting circuit behaviors,underscoring the potential of universal biological parts and predictive modeling in synthetic bioengineering.

A salt, detergent, and solvent tolerant protease from <i>Bacillus</i>sp. B001: Low-cost, easy-purified, and enhanced production by raw material based culture strategy

To enhance the protease production and decrease cost, corn flour and soy peptone were screened as cheap raw materials for the production of extracellular proteases by Bacillus strains. Their compositions in the medium suitable for enzyme production of Bacillus sp. B001 were optimized using statistical experiment designs. Under the optimized conditions, the protease production of Bacillus sp. B001 was stable at the stationary stage and reached to 63,200 U/mL, approximately 1.84-fold increase compared with that using the original medium. These improvements could be attributed to the release of the catabolite repression by crude materials corn flour and soy peptone which contained low level of available nutrients. Additionally, a highly pure protease which displayed excellent stability and compatibility with high salinity, commercial laundry detergents, and organic solvents, was rapidly obtained by two-step procedure involving ammonium sulphate precipitation and anion exchange from the fermentation cultures of B001 in the optimized medium. When the culture method applied to other Bacillus strains, their protease yields were all remarkably increased approximately 2.9 to 8.5 folds. In conclusion, a low-cost, easy-purified, and effective producing strategy using the cheap raw materials was developed here, representing a potential application for protease production in various Industrial processes.

Scientists Reveal Structural Mechanisms of Nivolumab-based PD-1-PD-L1 Blockade

Nivolumab is a commercial monoclonal antibody(mAb)which has been approved by US FDA in 2014 to treat melanoma,non-small cell lung cancer,etc.However,how this cancer drug works remains unclear.Researchers from Prof.YAN Jinghua’s and Prof.George F.Gao’s groups at the Institute of Microbiology,

Engineering Rubisco to enhance CO_(2) utilization

Ribulose-1,5-bisphosphate carboxylase/oxygenase(Rubisco)is a pivotal enzyme that mediates the fixation of CO_(2).As the most abundant protein on earth,Rubisco has a significant impact on global carbon,water,and nitrogen cycles.However,the significantly low carboxylation activity and competing oxygenase activity of Rubisco greatly impede high carbon fixation efficiency.This review first summarizes the current efforts in directly or indirectly modifying plant Rubisco,which has been challenging due to its high conservation and limitations in chloroplast transformation techniques.However,recent advancements in understanding Rubisco biogenesis with the assistance of chaperones have enabled successful heterologous expression of all Rubisco forms,including plant Rubisco,in microorganisms.This breakthrough facilitates the acquisition and evaluation of modified proteins,streamlining the measurement of their activity.Moreover,the establishment of a screening system in E.coli opens up possibilities for obtaining high-performance mutant enzymes through directed evolution.Finally,this review emphasizes the utilization of Rubisco in microorganisms,not only expanding their carbon-fixing capabilities but also holding significant potential for enhancing biotransformation processes.

Both structure and function of human monoclonal antibodies contribute to enhancement of Zika virus infectivity in vitro

Dear Editor,Antibody-dependent enhancement（ADE）has long been recognized for dengue virus（DENV）in vitro and in vivo.It is now clear that antibodies to DENV can also enhance Zika virus（ZIKV）infection in vitro,and vice versa（Dejnirattisai et al.,2016;Stettler et al.,2016）.The characteristics of enhancing antibodies,however,remain elusive.

Tricarbocyclic core formation of tyrosine-decahydrofluorenes implies a three-enzyme cascade with XenF-mediated sigmatropic rearrangement as a prerequisite

Tyrosine-decahydrofluorene derivatives feature a fused[6.5.6]tricarbocyclic core and a 13-membered para-cyclophane ether.Herein,we identified new xenoacremones A,B,and C(1-3)from the fungal strain Xenoacremonium sinensis ML-31 and elucidated their biosynthetic pathway using gene deletion in the native strain and heterologous expression in Aspergillus nidulans.The hybrid polyketide synthaseenonribosomal peptide synthetase(PKS-NRPS)XenE together with enoyl reductase XenG were confirmed to be responsible for the formation of the tyrosine-nonaketide skeleton.This skeleton was subsequently dehydrated by XenA to afford a pyrrolidinone moiety.XenF catalyzed a novel sigmatropic rearrangement to yield a key cyclohexane intermediate as a prerequisite for the formation of the multi-ring system.Subsequent oxidation catalyzed by XenD supplied the substrate for XenC to link the para-cyclophane ether,which underwent subsequent spontaneous Diels-Alder reaction to give the end products.Thus,the results indicated that three novel enzymes XenF,XenD,and XenC coordinate to assemble the[6.5.6]tricarbocyclic ring and para-cyclophane ether during biosynthesis of complex tyrosine-decahydrofluorene derivatives.

Reconstitution of biosynthetic pathway for mushroom-derived cyathane diterpenes in yeast and generation of new“non-natural”analogues

Mushroom-derived cyathane-type diterpenes possess unusual chemical skeleton and diverse bioactivities.To efficiently supply bioactive cyathanes for deep studies and explore their structural diversity,de novo synthesis of cyathane diterpenes in a geranylgeranyl pyrophosphate engineered Saccharomyces cerevisiae is investigated.Aided by homologous analyses,one new unclustered FAD-dependent oxidase EriM accounting for the formation of allyl aldehyde and three new NADP(H)-dependent reductases in the biosynthesis of cyathanes are identified and elucidated.By combinatorial biosynthetic strategy,S.cerevisiae strains generating twenty-two cyathane-type diterpenes,including seven"unnatural"cyathane xylosides(12,13,14a,14b,19,20,and 22)are established.Compounds 12-14,19,and 20 show significant neurotrophic effects on PC 12 cells in the dose of 6.3-25.0μmol/L.These studies provide new insights into the divergent biosynthesis of mushroom-originated cyathanes and a straightforward approach to produce bioactive cyathane-type diterpenes.

Reprogramming the endogenous type I CRISPR-Cas system for simultaneous gene regulation and editing in Haloarcula hispanica

The type I system is the most widely distributed CRISPR-Cas system identified so far.Recently,we have revealed the natural reprogramming of the type I CRISPR effector for gene regulation with a crRNA-resembling RNA in halophilic archaea.Here,we conducted a comprehensive study of the impact of redesigned crRNAs with different spacer lengths on gene regulation with the native type I-B CRISPR system in Haloarcula hispanica.When the spacer targeting the chromosomal gene was shortened from 36 to 28 bp,transformation efficiencies of the spacer-encoding plasmids were improved by over three orders of magnitude,indicating a significant loss of interference.However,by conducting whole-genome sequencing and measuring the growth curves of the hosts,we still detected DNA cleavage and its influence on cell growth.Intriguingly,when the spacer was shortened to 24 bp,the transcription of the target gene was downregulated to 10.80%,while both interference and primed adaptation disappeared.By modifying the lengths of the spacers,the expression of the target gene could be suppressed to varying degrees.Significantly,by designing crRNAs with different spacer lengths and targeting different genes,we achieved simultaneous gene editing(cdc6E)and gene regulation(crtB)for the first time with the endogenous type I CRISPR-Cas system.

Systematically investigating the key features of the DNase deactivated Cpf1 for tunable transcription regulation in prokaryotic cells

With a unique crRNA processing capability,the CRISPR associated Cpf1 protein holds great potential for multiplex gene regulation.Unlike the well-studied Cas9 protein,however,conversion of Cpf1 to a transcription regulator and its related properties have not been systematically explored yet.In this study,we investigated the mutation schemes and crRNA requirements for the DNase deactivated Cpf1(dCpf1).By shortening the direct repeat sequence,we obtained genetically stable crRNA co-transcripts and improved gene repression with multiplex targeting.A screen of diversity-enriched PAM library was designed to investigate the PAMdependency of gene regulation by dCpf1 from Francisella novicida and Lachnospiraceae bacterium.We found novel PAM patterns that elicited strong or medium gene repressions.Using a computational algorithm,we predicted regulatory outputs for all possible PAM sequences,which spanned a large dynamic range that could be leveraged for regulatory purposes.These newly identified features will facilitate the efficient design of CRISPR-dCpf1 based systems for tunable multiplex gene regulation.

Cross-species binding spectrum of ACE2 to SARS-CoV-2 RBD

Dear Editor,Severe acute respiratory syndrome coronavirus 2(SARSCo V-2)is the cause of COVID-19,which has posed a massive threat to human health,economy,and security worldwide(Bai et al.,2022).Genomic analysis of SARS-Co V-2 and related coronaviruses revealed that SARS-Co V and SARS-Co V-2likely had ancestors(Xu et al.,2020),which might originate in bats,followed by subsequent spread within intermediate hosts(spillover hosts)and then transmission to humans(Wang et al.,2021).

The identification of a CD47-blocking“hotspot”and design of a CD47/PD-L1 dual-specific antibody with limited hemagglutination

Dear Editor,By targeting the programmed cell death 1(PD-1)pathway with monoclonal antibodies(mAbs),immune checkpoint therapy(ICT)has achieved unprecedented clinical success in the treatment of multiple tumors.1,2 Cancer cells evade the host immune system via both the tolerance of T cells and functional suppression of innate immune cells.3 CD47 provides a“do not eat me”signal by binding to signal regulatory protein alpha(SIRPα)to prevent innate immune cells from attacking host cells.4 Recently,macrophages were found to restore antitumor reactivity by blocking the interaction between upregulated CD47 on tumor cells and SIRPαon innate immune cells.5 However,it remains unknown whether there are blocking“hotspots”on CD47 for mAb-based anti-CD47 therapy or additional blocking hotspot regions within CD47 for therapeutic mAb development.Although it is overexpressed on tumor cells,CD47 is also expressed in many normal cells,including red blood cells and platelets.6 Some validated CD47-blocking mAbs under clinical investigation induce hemagglutination and anemia.7 Thus,designing an engineered CD47-blocking antibody to exert a therapeutic effect with limited hemagglutination is needed.

Light-driven synthetic microbial consortia:playing with an oxygen dilemma

Background:Light-driven synthetic microbial consortia are composed of photoautotrophs and heterotrophs.They exhibited better performance in stability,robustness and capacity for handling complex tasks when comparing with axenic cultures.Different from general microbial consortia,the intrinsic property of photosynthetic oxygen evolution in light-driven synthetic microbial consortia is an important factor affecting the functions of the consortia.Results:In light-driven microbial consortia,the oxygen liberated by photoautotrophs will result in an aerobic environment,which exerts dual effects on different species and processes.On one hand,oxygen is favorable to the synthetic microbial consortia when they are used for wastewater treatment and aerobic chemical production,in which biomass accumulation and oxidized product formation will benefit from the high energy yield of aerobic respiration.On the other hand,the oxygen is harmful to the synthetic microbial consortia when they were used for anaerobic processes including biohydrogen production and bioelectricity generation,in which the presence of oxygen will deactivate some biological components and compete for electrons.Conclusions:Developing anaerobic processes in using light-driven synthetic microbial consortia represents a costeffective alternative for production of chemicals from carbon dioxide and light.Thus,exploring a versatile approach addressing the oxygen dilemma is essential to enable light-driven synthetic microbial consortia to get closer to practical applications.

Development of an activity-directed selection system enabled significant improvement of the carboxylation efficiency of Rubisco

Photosynthetic CO2 fixation is the ultimate source of organic carbon on earth and thus is essential for crop production and carbon sequestration, Ribulose-1,5-bis- phosphate carboxylase/oxygenase （Rubisco） catalyzes the first step of photosynthetic CO2 fixation. However, the extreme low carboxylation efficiency of Rubisco makes it the most attractive target for improving pho- tosynthetic efficiency. Extensive studies have focused on re-engineering a more efficient enzyme, but the effort has been impeded by the limited understanding of its structure-function relationships and the lack of an effi- cient selection system towards its activity. To address the unsuccessful molecular engineering of Rubisco, we developed an Escherichia coil-based activity-directed selection system which links the growth of host cell solely to the Rubisco activity therein. A Synechococcus sp. PCC7002 Rubisco mutant with E49V and D82G sub- stitutions in the small subunit was selected from a total of 15,000 mutants by one round of evolution. This mutant showed an 85% increase in specific carboxyla- tion activity and a 45% improvement in catalytic efficiency towards CO2. The small-subunit E49V mutation was speculated to influence holoenzyme catalysis through interaction with the large-subunit Q225. This interaction is conserved among various Rubisco from higher plants and Chlamydomonas reinhardtii. Knowledge of these might provide clues for engineering Rubisco from higher plants, with the potential of increasing the crop yield.

Crystal clear： visualizing the intervention mechanism of the PD-1/PD-L1 interaction by two cancer therapeutic monoclonal antibodies

Antibody-based PD-IIPD-L1 blockade therapies have taken center stage in immunotherapies for cancer, with multiple clinical successes. PD-1 signaling plays pivotal roles in tumor-driven T-cell dysfunction. In contrast to prior approaches to generate or boost tumor-specific T-cell responses, antibody-based PD-1/PD-L1 blockade targets tumor-induced T-cell defects and restores pre- existing T-cell function to modulate antitumor immunity. in this review, the fundamental knowledge on the expression regulations and inhibitory functions of PD-1 and the present understanding of antibody-based PD-1/ PD-L1 blockade therapies are briefly summarized. We then focus on the recent breakthrough work concerning the structural basis of the PD-IIPD-Ls interaction and how therapeutic antibodies, pembrolizumab targeting PD-1 and avelumab targeting PD-L1, compete with the binding of PD-1/PD-L1 to interrupt the PD-1/PD-L1 interaction. We believe that this structural informationwill benefit the design and improvement of therapeutic antibodies targeting PD-1 signaling.

Identification of a hotspot on PD-L1 for pH-dependent binding by monoclonal antibodies for tumor therapy

Dear Editor,Monoclonal antibody(mAb)-based tumor immune checkpoint therapy(ICT)has gained particular interest in recent years.1 The molecular basis of binding between mAbs and PD-1 or PD-L1 has been reported,providing clear information of the binding“hotspots”for mAbs.2,3 Tumor suppression efficacy of PD-L1 specific mAbs relies on not only the blocking of PD-1/PD-L1 interaction to restore T cell reactivity,but also Fc-mediated tumor cell cytotoxity.PD-L1 antibody drug conjugate(ADC)for selective chemo-guided immune modulation of tumor has also been developed which has shown promising tumor suppression potency.4 MAbs that could bind to antigen in a pH-dependent manner would improve recycling of the antibodies and engineered IL-6R mAbs with pH-dependent binding properties have displayed increased lysosomal delivery and therapeutic potency.5 However,no PD-L1 specific mAb with pH-dependent binding property has been reported,and whether the binding to a specific region on PD-L1 would induce pH-dependent interaction remains unknown.

Enzymatic clickable functionalization of peptides via computationally engineered peptide amidase

Directed peptides C-terminal modification enabled by the engineered biomolecular catalyst-peptide amidase 12 B has been achieved via computational protein engineering. The engineered enzyme exhibits great promising potential in the C-terminal modification of opioid peptides using prop-2-yn-1-amine(PYA) or prop-2-en-l-amine(PEA) as the nucleophile. A variety of opioid peptides could be readily functionalized at the C-terminal chain in high yield in a mild and selective manner. Notably, modified opioid peptides bearing alkynyl moiety could be further functionalized through well-established click reaction.

Systematic assessment of Pichia pastoris system for optimized b -galactosidase production

1.Introduction b-Galactosidase(commonly known as b-lactase;EC 3.2.1.23)is a multifunctional enzyme that can catalyze the hydrolysis of terminal non-reducing b-D-galactose residues in b-D-galactosides or transfer the galactosyl residue to saccharide acceptors to yield galactooligosaccharides(GOS).b-Galactosidase has a variety of applications in food and medical industries such as hydrolysis of lactose in milk,manufacture of galactooligosaccharides(GOS)and treatment of lactose malabsorption[1].Although b-galactosidase is an ubiquitous enzyme existing in plants,animals and microorganisms,only a few b-galactosidases from Kluveromyces lactis,Aspergillus niger and Aspergillus oryzae are regarded as safe for food related industry applications.

Fatty acid feedstocks enable a highly efficient glyoxylate-TCA cycle for high-yield production of β-alanine

Metabolic engineering to produce tricarboxylic acid(TCA)cycle-derived chemicals is usually associated with problems of low production yield and impaired cellular metabolism.In this work,we found that fatty acid(FA)feedstocks could enable high-yield production of TCA cycle-derived chemicals,while maintaining an efficient and balanced metabolic flux of the glyoxylate-TCA cycle,which is favorable for both product synthesis and cell growth.Here,we designed a novel synthetic pathway for production of β-alanine,an important TCA cycle-derived product,from FAs with a high theortecial yield of 1.391 g/g.By introducing panD,improving aspA,and knocking out iclR,glyoxylate shunt was highly activated in FAs and the yield of β-alanine reached 0.71 g/g from FAs,much higher than from glucose.Blocking the TCA cycle at icd/sucA/fumAC nodes could increase β-alanine yield in a flask cultivation,but severely reduced cell growth and FA utilization during fed-batch processes.Replenishing oxaloacetate by knocking out aspC and recovering fumAC could restore the growth and lead to a titer of 35.57 g/l.After relieving the oxidative stress caused by FA metabolism,β-alanine production could reach 72.05 g/l with a maximum yield of 1.24 g/g,about 86% of the theoretical yield.Our study thus provides a promising strategy for the production of TCA cycle-derived chemicals.