Multiplexed in-situ mutagenesis driven by a dCas12a-based dual-function base editor.

Mutagenesis driving genetic diversity is vital for understanding and engineering biological systems. However, the lack of effective methods to generate in-situ mutagenesis in multiple genomic loci combinatorially limits the study of complex biological functions. Here, we design and construct MultiduBE, a dCas12a-based multiplexed dual-function base editor, in an all-in-one plasmid for performing combinatorial in-situ mutagenesis. Two synthetic effectors, duBE-1a and duBE-2b, are created by amalgamating the functionalities of cytosine deaminase (from hAPOBEC3A or hAID*Δ ), adenine deaminase (from TadA9), and crRNA array processing (from dCas12a). Furthermore, introducing the synthetic separator Sp4 minimizes interference in the crRNA array, thereby facilitating multiplexed in-situ mutagenesis in both Escherichia coli and Bacillus subtilis. Guided by the corresponding crRNA arrays, MultiduBE is successfully employed for cell physiology reprogramming and metabolic regulation. A novel mutation conferring streptomycin resistance has been identified in B. subtilis and incorporated into the mutant strains with multiple antibiotic resistance. Moreover, surfactin and riboflavin titers of the combinatorially mutant strains improved by 42% and 15-fold, respectively, compared with the control strains with single gene mutation. Overall, MultiduBE provides a convenient and efficient way to perform multiplexed in-situ mutagenesis.

A MOF-Gel Based Separator for Suppressing Redox Mediator Shuttling in Li-O2 Batteries.

Redox mediators (RMs) are widely utilized in the electrolytes of Li-O2 batteries to catalyze the formation/decomposition of Li2O2, which significantly enhances the cycling performance and reduces the charge overpotential. However, RMs have a shuttle effect by migrating to the Li anode side and inducing Li metal degradation through a parasitic reaction. Herein, a metal-organic framework gel (MOF-gel) separator is proposed to restrain the shuttling of RMs. Compared to traditional MOF nanoparticles, MOF gels form uniform and dense films on the separators. When using Ru(acac)3 (ruthenium acetylacetonate) as an RM, the MOF-gel separator suppresses the shuttling of Ru(acac)3 toward the Li anode side and significantly enhances the performance of Li-O2 batteries. Specifically, Li-O2 batteries exhibit an ultralong cycling life (410 cycles) at a current density of 0.5 A g-1. Moreover, the batteries using the MOF-gel/celgard separator exhibit significantly improved cycling performance (increase by ≈1.6 times) at a high current density of 1.0 A g-1 and a decreased charge/discharge overpotential. This result is expected to guide future development of battery separators and the exploration of redox mediators.

AcrR1, a novel TetR/AcrR family repressor, mediates acid and antibiotic resistance and nisin biosynthesis in Lactococcus lactis F44.

Lactococcus lactis, widely used in the manufacture of dairy products, encounters various environmental stresses both in natural habitats and during industrial processes. It has evolved intricate machinery of stress sensing and defense to survive harsh stress conditions. Here, we identified a novel TetR/AcrR family transcription regulator, designated AcrR1, to be a repressor for acid and antibiotic tolerance that was derepressed in the presence of vancomycin or under acid stress. The survival rates of acrR1 deletion strain ΔAcrR1 under acid and vancomycin stresses were about 28.7-fold (pH 3.0, HCl), 8.57-fold (pH 4.0, lactic acid) and 2.73-fold (300 ng/mL vancomycin) as that of original strain F44. We also demonstrated that ΔAcrR1 was better able to maintain intracellular pH homeostasis and had a lower affinity to vancomycin. No evident effects of AcrR1 deletion on the growth and morphology of strain F44 were observed. Subsequently, we characterized that the transcription level of genes associated with amino acids biosynthesis, carbohydrate transport and metabolism, multiple drug resistance and DNA repair proteins significantly upregulated in ΔAcrR1 using transcriptome analysis and quantitative reverse transcription-PCR (qRT-PCR) assays. Additionally, AcrR1 could repress the transcription of nisin post-translational modification gene, nisC, leading to a 16.3% increase in nisin yield after AcrR1 deletion. Our results not only refined the knowledge of the regulatory mechanism of TetR/AcrR family regulator in L. lactis, but presented a potential strategy to enhance industrial production of nisin.

Incorporation of Non-Canonical Amino Acids into Antimicrobial Peptides: Advances, Challenges, and Perspectives.

The emergence of antimicrobial resistance is a global health concern and calls for the development of novel antibiotic agents. Antimicrobial peptides seem to be promising candidates due to their diverse sources, mechanisms of action, and physicochemical characteristics, as well as the relatively low emergence of resistance. The incorporation of noncanonical amino acids into antimicrobial peptides could effectively improve their physicochemical and pharmacological diversity. Recently, various antimicrobial peptides variants with improved or novel properties have been produced by the incorporation of single and multiple distinct noncanonical amino acids. In this review, we summarize strategies for the incorporation of noncanonical amino acids into antimicrobial peptides, as well as their features and suitabilities. Recent applications of noncanonical amino acid incorporation into antimicrobial peptides are also presented. Finally, we discuss the related challenges and prospects.

Combinational quorum sensing devices for dynamic control in cross-feeding cocultivation.

Quorum sensing (QS) offers cell density dependent dynamic regulations in cell culture through devices such as synchronized lysis circuit (SLC) and metabolic toggle switch (MTS). However, there is still a lack of studies on cocultivation with a combination of different QS-based devices. Taking the production of isopropanol and salidroside as case studies, we have mathematically modeled a comprehensive set of QS-regulated cocultivation schemes and constructed experimental combinations of QS devices, respectively, to evaluate their feasibility and optimality for regulating growth competition and corporative production. Glucose split ratio is proposed for the analysis of competition between cell growth and targeted production. Results show that the combination of different QS devices across multiple members offers a new tool with the potential to effectively coordinate synthetic microbial consortia for achieving high product titer in cross-feeding cocultivation. It is also evident that the performance of such systems is significantly affected by dynamic characteristics of chosen QS devices, carbon source control and the operational settings. This study offers insights for future applications of combinational QS devices in synthetic microbial consortia.

Quorum sensing for population-level control of bacteria and potential therapeutic applications.

Quorum sensing (QS), a microbial cell-to-cell communication process, dynamically regulates a variety of metabolism and physiological activities. In this review, we provide an update on QS applications based on autoinducer molecules including acyl-homoserine lactones (AHLs), auto-inducing peptides (AIPs), autoinducer 2 (AI-2) and indole in population-level control of bacteria, and highlight the potential in developing novel clinical therapies. We summarize the development in the combination of various genetic circuits such as genetic oscillators, toggle switches and logic gates with AHL-based QS devices in Gram-negative bacteria. An overview is then offered to the state-of-the-art of much less researched applications of AIP-based QS devices with Gram-positive bacteria, followed by a review of the applications of AI-2 and indole based QS for interspecies communication among microbial communities. Building on these general-purpose QS applications, we highlight the disruptions and manipulations of QS devices as potential clinical therapies for diseases caused by biofilm formation, antibiotic resistance and the phage invasion. The last part of reviewed literature is dedicated to mathematical modelling for QS applications. Finally, the key challenges and future perspectives of QS applications in monoclonal synthetic biology and synthetic ecology are discussed.

NisI Maturation and Its Influence on Nisin Resistance in Lactococcus lactis.

NisI confers immunity against nisin, with high substrate specificity to prevent a suicidal effect in nisin-producing Lactococcus lactis strains. However, the NisI maturation process as well as its influence on nisin resistance has not been characterized. Here, we report the roles of lipoprotein signal peptidase II (Lsp) and prolipoprotein diacylglyceryl transferase (Lgt) in NisI maturation and nisin resistance of L. lactis F44. We found that the resistance of nisin of an Lsp-deficient mutant remarkably decreased, while no significant differences in growth were observed. We demonstrated that Lsp could cleave signal peptide of NisI precursor in vitro Moreover, diacylglyceryl modification of NisI catalyzed by Lgt played a decisive role in attachment of NisI on the cell envelope, while it exhibited no effects on cleavage of the signal peptides of NisI precursor. The dissociation constant (KD ) for the interaction between nisin and NisI exhibited a 2.8-fold increase compared with that between nisin and pre-NisI with signal peptide by surface plasmon resonance (SPR) analysis, providing evidence that Lsp-catalyzed signal peptide cleavage was critical for the immune activity of NisI. Our study revealed the process of NisI maturation in L. lactis and presented a potential strategy to enhance industrial nisin production.IMPORTANCE Nisin, a safe and natural antimicrobial peptide, has a long and impressive history as a food preservative and is also considered a novel candidate to alleviate the increasingly serious threat of antibiotic resistance. Nisin is produced by certain L. lactis strains. The nisin immunity protein NisI, a membrane-bound lipoprotein, is expressed by nisin producers to avoid suicidal action. Here, we report the roles of Lsp and Lgt in NisI maturation and nisin resistance of L. lactis F44. The results verified the importance of Lsp to NisI-conferred immunity and Lgt to localization. Our study revealed the process of NisI maturation in L. lactis and presented a potential strategy to enhance industrial nisin production.

Whole-Genome-Based Survey for Polyphyletic Serovars of Salmonella enterica subsp. enterica Provides New Insights into Public Health Surveillance.

Serotyping has traditionally been considered the basis for surveillance of Salmonella, but it cannot distinguish distinct lineages sharing the same serovar that vary in host range, pathogenicity and epidemiology. However, polyphyletic serovars have not been extensively investigated. Public health microbiology is currently being transformed by whole-genome sequencing (WGS) data, which promote the lineage determination using a more powerful and accurate technique than serotyping. The focus in this study is to survey and analyze putative polyphyletic serovars. The multi-locus sequence typing (MLST) phylogenetic analysis identified four putative polyphyletic serovars, namely, Montevideo, Bareilly, Saintpaul, and Muenchen. Whole-genome-based phylogeny and population structure highlighted the polyphyletic nature of Bareilly and Saintpaul and the multi-lineage nature of Montevideo and Muenchen. The population of these serovars was defined by extensive genetic diversity, the open pan genome and the small core genome. Source niche metadata revealed putative existence of lineage-specific niche adaptation (host-preference and environmental-preference), exhibited by lineage-specific genomic contents associated with metabolism and transport. Meanwhile, differences in genetic profiles relating to virulence and antimicrobial resistance within each lineage may contribute to pathogenicity and epidemiology. The results also showed that recombination events occurring at the H1-antigen loci may be an important reason for polyphyly. The results presented here provide the genomic basis of simple, rapid, and accurate identification of phylogenetic lineages of these serovars, which could have important implications for public health.

Two-stage carbon distribution and cofactor generation for improving l-threonine production of Escherichia coli.

L-Threonine, a kind of essential amino acid, has numerous applications in food, pharmaceutical, and aquaculture industries. Fermentative l-threonine production from glucose has been achieved in Escherichia coli. However, there are still several limiting factors hindering further improvement of l-threonine productivity, such as the conflict between cell growth and production, byproduct accumulation, and insufficient availability of cofactors (adenosine triphosphate, NADH, and NADPH). Here, a metabolic modification strategy of two-stage carbon distribution and cofactor generation was proposed to address the above challenges in E. coli THRD, an l-threonine producing strain. The glycolytic fluxes towards tricarboxylic acid cycle were increased in growth stage through heterologous expression of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and citrate synthase, leading to improved glucose utilization and growth performance. In the production stage, the carbon flux was redirected into l-threonine synthetic pathway via a synthetic genetic circuit. Meanwhile, to sustain the transaminase reaction for l-threonine production, we developed an l-glutamate and NADPH generation system through overexpression of glutamate dehydrogenase, formate dehydrogenase, and pyridine nucleotide transhydrogenase. This strategy not only exhibited 2.02- and 1.21-fold increase in l-threonine production in shake flask and bioreactor fermentation, respectively, but had potential to be applied in the production of many other desired oxaloacetate derivatives, especially those involving cofactor reactions.

A novel small RNA S042 increases acid tolerance in Lactococcus lactis F44.

Lactococcus lactis, a gram-positive bacterium, encounters various environmental stresses, especially acid stress, during fermentation. Small RNAs (sRNAs) that serve as regulators at post-transcriptional level play important roles in acid stress response. Here, a novel sRNA S042 was identified by RNA-Seq, RT-PCR and Northern blot. The transcription level of s042 was upregulated 2.29-fold under acid stress by Quantitative RT-PCR (qRT-PCR) analysis. Acid tolerance assay showed that overexpressing s042 increased the survival rate of L. lactis F44 and deleting s042 significantly inhibited the viability under acidic conditions. Moreover, the targets were predicted by online software and four genes were chosen as candidates. Among them, argR (arginine regulator) and accD (acetyl-CoA carboxylase carboxyl transferase subunit beta) were validated to be the direct targets activated by S042 through reporter fusion assay. The regulatory mechanism between S042 and its targets was further investigated through Bioinformatics and qRT-PCR. This study served to highlight the role of the novel sRNA S042 in acid resistance of L. lactis and provided new insights into the response mechanism of acid stress.

Global evolution of glycosylated polyene macrolide antibiotic biosynthesis.

Antibiotics are the most marvelous evolutionary products of microbes to obtain competitive advantage and maintain ecological balance. However, the origination and development of antibiotics has yet to be explicitly investigated. Due to diverse structures and similar biosynthesis, glycosylated polyene macrolides (gPEMs) were chosen to explore antibiotic evolution. A total of 130 candidate and 38 transitional gPEM clusters were collected from actinomycetes genomes, providing abundant references for phenotypic gaps in gPEM evolution. The most conserved parts of gPEM biosynthesis were found and used for phylogeny construction. On this basis, we proposed ancestral gPEM clusters at different evolutionary stages and interpreted the possible evolutionary histories in detail. The results revealed that gPEMs evolved from small rings to large rings and continuously increased structural diversity through acquiring, discarding and exchanging genes from different evolutionary origins, as well as co-evolution of functionally related proteins. The combination of horizontal gene transfers, environmental effects and host preference resulted in the diversity and worldwide distribution of gPEMs. This study is not only a useful exploration on antibiotic evolution but also an inspiration for diversity and biogeographic investigations on antibiotics in the era of Big Data.

Redox cofactor engineering in industrial microorganisms: strategies, recent applications and future directions.

NAD and NADP, a pivotal class of cofactors, which function as essential electron donors or acceptors in all biological organisms, drive considerable catabolic and anabolic reactions. Furthermore, they play critical roles in maintaining intracellular redox homeostasis. However, many metabolic engineering efforts in industrial microorganisms towards modification or introduction of metabolic pathways, especially those involving consumption, generation or transformation of NAD/NADP, often induce fluctuations in redox state, which dramatically impede cellular metabolism, resulting in decreased growth performance and biosynthetic capacity. Here, we comprehensively review the cofactor engineering strategies for solving the problematic redox imbalance in metabolism modification, as well as their features, suitabilities and recent applications. Some representative examples of in vitro biocatalysis are also described. In addition, we briefly discuss how tools and methods from the field of synthetic biology can be applied for cofactor engineering. Finally, future directions and challenges for development of cofactor redox engineering are presented.

Glycosyltransferases: mechanisms and applications in natural product development.

Glycosylation reactions mainly catalyzed by glycosyltransferases (Gts) occur almost everywhere in the biosphere, and always play crucial roles in vital processes. In order to understand the full potential of Gts, the chemical and structural glycosylation mechanisms are systematically summarized in this review, including some new outlooks in inverting/retaining mechanisms and the overview of GT-C superfamily proteins as a novel Gt fold. Some special features of glycosylation and the evolutionary studies on Gts are also discussed to help us better understand the function and application potential of Gts. Natural product (NP) glycosylation and related Gts which play important roles in new drug development are emphasized in this paper. The recent advances in the glycosylation pattern (particularly the rare C- and S-glycosylation), reversibility, iterative catalysis and protein auxiliary of NP Gts are all summed up comprehensively. This review also presents the application of NP Gts and associated studies on synthetic biology, which may further broaden the mind and bring wider application prospects.

Immobilization of Streptomyces thermotolerans 11432 on polyurethane foam to improve production of acetylisovaleryltylosin.

In this study, polyurethane foam (PUF) was chemically treated to immobilize Streptomyces thermotolerans 11432 for semi-continuous production of acetylisovaleryltylosin (AIV). Based on experimental results, positive cross-linked PUF (PCPUF) was selected as the most effective carrier according to immobilized cell mass. The effect of adsorption time on immobilized mass was investigated. AIV concentration (33.54 mg/l) in batch fermentations with immobilized cells was higher than with free cells (20.34 mg/l). In repeated batch fermentations with immobilized S. thermotolerans 11432 using PCPUF cubes, high AIV concentrations and conversion rates were attained, ranging from 25.56 to 34.37 mg/l and 79.93 to 86.31 %, respectively. Significantly, this method provides a feasible strategy for efficient AIV production and offers the potential for large-scale production.

[Knockdown of motility-related genes of Komagataeibacter xylinus and its effect on bacterial cellulose synthesis].

Bacterial cellulose (BC) is a biopolymer synthesized by bacteria, which possess excellent characteristics such as high water holding capacity, high crystallinity, and high purity. It is widely used in food, medical, cosmetics, and functional films. Komagataeibacter xylinus is a model strain used in BC synthesis research. In bacteria, motility-related genes are associated with BC synthesis, whereas in Komagataeibacter xylinus CGMCC 2955, the functions of motility-related genes and their effects on BC synthesis are not known. To address this gap, we used the λ Red recombinant system to individually knock out motA, motB, and mot2A respectively, and constructed the knockout strains K. x-ΔmotA, K. x-ΔmotB, and K. x-Δmot2A. Additionally, both motA and motB were disrupted to construct the K. x-ΔmotAB mutant. The results demonstrated that knockout strain K. x-ΔmotAB exhibited the highest BC yield, reaching (5.05±0.26) g/L, which represented an increase of approximately 24% compared to wild-type strains. Furthermore, the BC synthesized by this strain exhibited the lowest porosity, 54.35%, and displayed superior mechanical properties with a Young's modulus of up to 5.21 GPa. As knocking out motA and motB genes in K. xylinus CGMCC 2955 did not reduce BC yield; instead, it promoted BC synthesis. Consequently, this research further deepened our understanding of the relationship between motility and BC synthesis in acetic acid bacteria. The knockouts of motA and motB genes resulted in reduced BC porosity and improved mechanical properties, provides a reference for BC synthesis and membrane structure regulation modification.

In Silico Prediction and Mining of Exporters for Secretory Bioproduction of Terpenoids in Saccharomyces cerevisiae.

Terpenoids are the largest class of natural products, and their bioproduction by engineered cell factories receives high attention. However, excessive intracellular accumulation is one of the bottlenecks that limit the further improvement of the yield of terpenoid products. Therefore, it is important to mine exporters to achieve the secretory production of terpenoids. This study proposed a framework for the in silico prediction and mining of terpenoid exporters in Saccharomyces cerevisiae. Through the process of "mining-docking-construction-validation", we found that Pdr5 of ATP-binding cassette (ABC) transporters and Osh3 of oxysterol-binding homology (Osh) proteins can promote squalene efflux. Squalene secretion of the strain overexpressing Pdr5 and Osh3 increased to 141.1 times that of the control strain. Besides squalene, ABC exporters also can promote the secretion of ß-carotene and retinal. Molecular dynamics simulation results revealed that before exporter conformations transitioned to the "outward-open" states, the substrates might have bound to the tunnels and prepared for rapid efflux. Overall, this study provides a terpenoid exporter prediction and mining framework that may be generally used to identify exporters of other terpenoids.

Metabolic Engineering of Saccharomyces cerevisiae for Efficient Retinol Synthesis.

Retinol, the main active form of vitamin A, plays a role in maintaining vision, immune function, growth, and development. It also inhibits tumor growth and alleviates anemia. Here, we developed a Saccharomyces cerevisiae strain capable of high retinol production. Firstly, the de novo synthesis pathway of retinol was constructed in S. cerevisiae to realize the production of retinol. Second, through modular optimization of the metabolic network of retinol, the retinol titer was increased from 3.6 to 153.6 mg/L. Then, we used transporter engineering to regulate and promote the accumulation of the intracellular precursor retinal to improve retinol production. Subsequently, we screened and semi-rationally designed the key enzyme retinol dehydrogenase to further increase the retinol titer to 387.4 mg/L. Lastly, we performed two-phase extraction fermentation using olive oil to obtain a final shaking flask retinol titer of 1.2 g/L, the highest titer reported at the shake flask level. This study laid the foundation for the industrial production of retinol.

Combinatorial metabolic engineering enables the efficient production of ursolic acid and oleanolic acid in Saccharomyces cerevisiae.

Ursolic acid (UA) and oleanolic acid (OA) have been demonstrated to have promising therapeutic potential as anticancer and bacteriostasis agents. Herein, via the heterologous expression and optimization of CrAS, CrAO, and AtCPR1, the de novo syntheses of UA and OA were achieved with titers of 7.4 and 3.0 mg/L, respectively. Subsequently, metabolic flux was redirected by increasing the cytosolic acetyl-CoA level and tuning the copy numbers of ERG1 and CrAS, thereby affording 483.4 mg/L UA and 163.8 mg/L OA. Furthermore, the lipid droplet compartmentalization of CrAO and AtCPR1 alongside the strengthening of the NADPH regeneration system increased the UA and OA titers to 692.3 and 253.4 mg/L in a shake flask and to 1132.9 and 433.9 mg/L in a 3-L fermenter, which is the highest UA titer reported to date. Overall, this study provides a reference for constructing microbial cell factories that can efficiently synthesize terpenoids.

High-Rate Lithium-Selenium Batteries Boosted by a Multifunctional Janus Separator Over a Wide Temperature Range of -30 °C to 60 °C.

Lithium-selenium batteries are characterized by high volumetric capacity comparable to Li-S batteries, while ≈1025 times higher electrical conductivity of Se than S is favorable for high-rate capability. However, they also suffer from the "shuttling effect" of lithium polyselenides (LPSes) and Li dendrite growth. Herein, a multifunctional Janus separator is designed by coating hierarchical nitrogen-doped carbon nanocages (hNCNC) and AlN nanowires on two sides of commercial polypropylene (PP) separator to overcome these hindrances. At room temperature, the Li-Se batteries with the Janus separator exhibit an unprecedented high-rate capability (331 mAh g-1 at 25 C) and retain a high capacity of 408 mAh g-1 at 3 C after 500 cycles. Moreover, the high retained capacities are achieved over a wide temperature range from -30 °C to 60 °C, showing the potential application under extreme environments. The excellent performances result from the "1+1>2" synergism of suppressed LPSes shuttling by chemisorption and electrocatalysis of hNCNC on the cathode side and suppressed Li-dendrite growth by thermally conductive AlN-network on the anode side, which can be well understood by the "Bucket Effect". This Janus separator provides a general strategy to develop high-performance lithium-chalcogen (Se, S, SeS2 ) batteries.

APSNet: Toward Adaptive Point Sampling for Efficient 3D Action Recognition.

Observing that it is still a challenging task to deploy 3D action recognition methods in real-world scenarios, in this work, we investigate the accuracy-efficiency trade-off for 3D action recognition. We first introduce a simple and efficient backbone network structure for 3D action recognition, in which we directly extract the geometry and motion representations from the raw point cloud videos through a set of simple operations (i.e., coordinate offset generation and mini-PoinNet). Based on the backbone network, we propose an end-to-end optimized network called adaptive point sampling network (APSNet) to achieve the accuracy-efficiency trade-off, which mainly consists of three stages: the coarse feature extraction stage, the decision making stage, and the fine feature extraction stage. In APSNet, we adaptively decide the optimal resolutions (i.e., the optimal number of points) for each pair of frames based on any input point cloud video under the given computational complexity constraint. Comprehensive experiments on multiple benchmark datasets demonstrate the effectiveness and efficiency of our newly proposed APSNet for 3D action recognition.