A self-regulated network for dynamically balancing multiple precursors in complex biosynthetic pathways.

Microbial synthesis has emerged as a promising and sustainable alternative to traditional chemical synthesis and plant extraction. However, the competition between synthetic pathways and central metabolic pathways for cellular resources may impair final production efficiency. Moreover, when the synthesis of target product requires multiple precursors from the same node, the conflicts of carbon flux have further negative impacts on yields. In this study, a self-regulated network was developed to relieve the competition of precursors in complex synthetic pathways. Using 4-hydroxycoumarin (4-HC) synthetic pathway as a proof of concept, we employed an intermediate as a trigger to dynamically rewire the metabolic flux of pyruvate and control the expression levels of genes in 4-HC synthetic pathway, achieving self-regulation of multiple precursors and enhanced titer. Transcriptomic analysis results additionally demonstrated that the gene transcriptional levels of both pyruvate kinase PykF and synthetic pathway enzyme SdgA dynamically changed according to the intermediate concentrations. Overall, our work established a self-regulated network to dynamically balance the metabolic flux of two precursors in 4-HC biosynthesis, providing insight into balancing biosynthetic pathways where multiple precursors compete and interfere with each other.

CmERF5-CmRAP2.3 transcriptional cascade positively regulates waterlogging tolerance in Chrysanthemum morifolium.

Waterlogging stress affects plant growth by limiting root respiration and reducing yield and economic value. Therefore, identifying genes involved in regulating waterlogging stress is vital. This study reports the ethylene-responsive VII transcription factor (CmRAP2.3) in the chrysanthemum. Subcellular localization and transactivation assay analyses revealed that CmRAP2.3 was localized in the nucleus and possessed transactivation activity. Overexpression of CmRAP2.3 in chrysanthemum was found to enhance waterlogging tolerance by decreasing reactive oxygen species (ROS) levels. Furthermore, we found that the transcription factor CmERF5 binds to GCC-like motifs in the CmRAP2.3 promoter region and activates CmRAP2.3 expression. Additionally, CmERF5 overexpression maintained a low ROS level and improved chrysanthemum waterlogging tolerance. Taken together, this study shows a molecular mechanism by which CmERF5 transcriptionally activates CmRAP2.3 to reduce waterlogging stress via the ROS pathway in the chrysanthemum.

Biosensor-assisted titratable CRISPRi high-throughput (BATCH) screening for over-production phenotypes.

With rapid advances in the development of metabolic pathways and synthetic biology toolkits, a persisting challenge in microbial bioproduction is how to optimally rewire metabolic fluxes and accelerate the concomitant high-throughput phenotype screening. Here we developed a biosensor-assisted titratable CRISPRi high-throughput (BATCH) screening approach that combines a titratable mismatch CRISPR interference and a biosensor mediated screening for high-production phenotypes in Escherichia coli. We first developed a programmable mismatch CRISPRi that could afford multiple levels of interference efficacy with a one-pot sgRNA pool (a total of 16 variants for each target gene) harboring two consecutive random mismatches in the seed region of sgRNA spacers. The mismatch CRISPRi was demonstrated to enable almost a full range of gene knockdown when targeting different positions on genes. As a proof-of-principle demonstration of the BATCH screening system, we designed doubly mismatched sgRNA pools targeting 20 relevant genes in E. coli and optimized a PadR-based p-coumaric acid biosensor with broad dynamic range for the eGFP fluorescence guided high-production screening. Using sgRNA variants for the combinatorial knockdown of pfkA and ptsI, the p-coumaric acid titer was increased by 40.6% to o 1308.6 mg/l from glycerol in shake flasks. To further demonstrate the general applicability of the BATCH screening system, we recruited a HpdR-based butyrate biosensor that facilitated the screening of E. coli strains achieving 19.0% and 25.2% increase of butyrate titer in shake flasks with sgRNA variants targeting sucA and ldhA, respectively. This work reported the establishment of a plug-and-play approach that enables multilevel modulation of metabolic fluxes and high-throughput screening of high-production phenotypes.

Exploring and engineering PAM-diverse Streptococci Cas9 for PAM-directed bifunctional and titratable gene control in bacteria.

The RNA-guided Cas9s serve as powerful tools for programmable gene editing and regulation; their targeting scopes and efficacies, however, are always constrained by the PAM sequence stringency. Most Streptococci Cas9s, including the prototype SpCas9 from S. pyogenes, specifically recognize a canonical NGG PAM via a conserved RxR PAM-binding motif within the PAM-interaction (PI) domain. Here, SpCas9-based mining unveils three distinct and rarely presented PAM-binding motifs (QxxxR, QxQ and RxQ) among Streptococci Cas9 orthologs. With the catalytically-dead QxxxR-containing SedCas9 from S. equinus, we dissect its NAG PAM specificity and elucidate its underlying recognition mechanism via computational prediction and mutagenesis analysis. Replacing the SedCas9 PI domain with alternate PAM-binding motifs rewires its PAM specificity to NGG or NAA. Moreover, a semi-rational design with minimal mutation creates a SedCas9-NQ variant showing robust activity towards expanded NNG and NAA PAMs, based upon which we engineered a compact ω-SedCas9-NQ transcriptional regulator for PAM-directed bifunctional and titratable gene control. The ω-SedCas9-NQ mediated metabolic reprogramming of endogenous genes in Escherichia coli affords a 2.6-fold increase of 4-hydroxycoumarin production. This work reveals new Cas9 scaffolds with distinct PAM-binding motifs for PAM relaxation and creates a new PAM-diverse Cas9 variant for versatile gene control in bacteria.

Establishing biosynthetic pathway for the production of p-hydroxyacetophenone and its glucoside in Escherichia coli.

p-Hydroxyacetophenone (p-HAP) and its glucoside picein are plant-derived natural products that have been extensively used in chemical, pharmaceutical and cosmetic industries owing to their antioxidant, antibacterial and antiseptic activities. However, the natural biosynthetic pathways for p-HAP and picein have yet been resolved so far, limiting their biosynthesis in microorganisms. In this study, we design and construct a biosynthetic pathway for de novo production of p-HAP and picein from glucose in E. coli. First, screening and characterizing pathway enzymes enable us to successfully establish functional biosynthetic pathway for p-HAP production. Then, the rate-limiting step in the pathway caused by a reversible alcohol dehydrogenase is completely eliminated by modulating intracellular redox cofactors. Subsequent host strain engineering via systematic increase of precursor supplies enables production enhancement of p-HAP with a titer of 1445.3 mg/L under fed-batch conditions. Finally, a novel p-HAP glucosyltransferase capable of generating picein from p-HAP is identified and characterized from a series of glycosyltransferases. On this basis, de novo biosynthesis of picein from glucose is achieved with a titer of 210.7 mg/L under fed-batch conditions. This work not only demonstrates a microbial platform for p-HAP and picein synthesis, but also represents a generalizable pathway design strategy to produce value-added compounds.

Engineering Escherichia coli native metabolism for efficient biosynthesis of orotate.

Orotate (OA) is a precursor of pyrimidine nucleotides and is widely used in food, pharmaceutical, and cosmetic industries. Although various microorganisms have been used for OA production, the production efficiency needs to be further improved for industrial application. In this study, we engineered Escherichia coli native metabolism for efficient OA production. The entire pathway was divided into the downstream OA synthesis, the midstream aspartate/glutamine supply, and the upstream glycolysis modules. First, the downstream module was optimized by disrupting pyrE to block OA consumption and release the feedback inhibition, and tuning expression of the biosynthetic genes. Second, the midstream pathway was enhanced by increasing the supply of the precursors and the cofactor nicotinamide adenine dinucleotide phosphate (NADPH). More importantly, we observed that pyrE disruption may lead to metabolic disorder as indicated by the accumulation of large amount of acetate. This problem was solved by reducing the flux of glycolysis. With these efforts, the final strain produced 80.3 g/L OA with a yield of 0.56 g/g glucose in fed-batch fermentation, which are the highest titer and yield reported so far. This work paves the way for industrial production of OA and represents as a good example of modulating cell metabolism for efficient chemical production.

Adverse pregnancy outcomes among pregnant women living with HIV in Hubei province, China: prevalence and risk factors.

Mother-to-child transmission of Human Immunodeficiency Virus (HIV) has been greatly reduced with the advance of intervention technology. However, adverse pregnancy outcomes (APOs) are still common, and little is known about the driving forces of APOs among pregnant women living with HIV in China. Between January 2004 and December 2020, a total of 638 pregnancies among pregnant women living with HIV were enrolled in this study, 84 (13.2%) pregnancies with 87 APOs were reported. Preterm birth (3.8%), ectopic pregnancy (3.4%), spontaneous abortion (2.0%), and embryo arrest (1.7%) were the most common APOs in pregnant women living with HIV. Exposure to antiretroviral drugs (ARVs) during the first trimester (RR = 4.077, 95% CI: 0.521, 1.484, P<0.001) and the first CD4+ T lymphocyte count (CD4 count)≤ 350/µl (RR = 2.227, 95% CI: 0.063, 0.991, P = 0.026) were risk factors of APOs. The age≤ 30 years (RR = -2.513, 95% CI: -1.067, -0.132, P = 0.012) was associated with the decreasing of APOs. Encouraging people to initiate combination antiretroviral therapy and reach a high CD4 count level before pregnancy would be helpful to prevent APOs. Pregnant women exposed to ARVs in the first trimester needed more attention for APOs.

Bacterial synthesis of C3-C5 diols via extending amino acid catabolism.

Amino acids are naturally occurring and structurally diverse metabolites in biological system, whose potentials for chemical expansion, however, have not been fully explored. Here, we devise a metabolic platform capable of producing industrially important C3-C5 diols from amino acids. The presented platform combines the natural catabolism of charged amino acids with a catalytically efficient and thermodynamically favorable diol formation pathway, created by expanding the substrate scope of the carboxylic acid reductase toward noncognate ω-hydroxylic acids. Using the established platform as gateways, seven different diol-convertible amino acids are converted to diols including 1,3-propanediol, 1,4-butanediol, and 1,5-pentanediol. Particularly, we afford to optimize the production of 1,4-butanediol and demonstrate the de novo production of 1,5-pentanediol from glucose, with titers reaching 1.41 and 0.97 g l-1, respectively. Our work presents a metabolic platform that enriches the pathway repertoire for nonnatural diols with feedstock flexibility to both sugar and protein hydrolysates.

Harnessing plasmid replication mechanism to enable dynamic control of gene copy in bacteria.

Dynamic regulation has been proved efficient in controlling gene expression at transcriptional, translational, and post-translational level. However, the dynamic regulation at gene replication level has been rarely explored so far. In this study, we established dynamic regulation at gene copy level through engineering controllable plasmid replication to dynamically control the gene expression. Prototypic genetic circuits with different control logic were applied to enable diversified dynamic behaviors of gene copy. To explore the applicability of this strategy, the dynamic gene copy control was employed in regulating the biosynthesis of p-coumaric acid, which resulted in an up to 78% increase in p-coumaric acid titer to 1.69 g/L in shake flasks. These results indicated the great potential of applying dynamic gene copy control for engineering biosynthesis of valuable compounds in metabolic engineering.

Establishing an Autonomous Cascaded Artificial Dynamic (AutoCAD) regulation system for improved pathway performance.

Endogenous metabolic pathways in microbial cells are usually precisely controlled by sophisticated regulation networks. However, the lack of such regulations when introducing heterologous pathways in microbial hosts often causes unbalanced enzyme expression and carbon flux distribution, hindering the construction of highly efficient microbial biosynthesis systems. Here, using naringenin as the target compound, we developed an Autonomous Cascaded Artificial Dynamic (AutoCAD) regulation system to automatically coordinate the pathway expression and redirect carbon fluxes for enhanced naringenin production. The AutoCAD regulation system, consisting of both intermediate-based feedforward and product-based feedback control genetic circuits, resulted in a 16.5-fold increase in naringenin titer compared with the static control. Fed-batch fermentation using the strain with AutoCAD regulation further enhanced the naringenin titer to 277.2 mg/L. The AutoCAD regulation system, with intermediate-based feedforward control and product-triggered feedback control, provides a new paradigm of developing complicated cascade dynamic control to engineer heterologous pathways.

Targeting cofactors regeneration in methylation and hydroxylation for high level production of Ferulic acid.

Ferulic acid (FA) is a natural methylated phenolic acid which represents various bioactivities. Bioproduction of FA suffers from insufficient methyl donor supplement and inefficient hydroxylation. To overcome these hurdles, we first activate the S-adenosylmethionine (SAM) cycle in E. coli by using endogenous genes to supply sufficient methyl donor. Then, a small protein Fre is introduced into the pathway to efficiently regenerate FADH2 for the hydroxylation. Remarkably, regeneration of these two cofactors dramatically promotes FA synthesis. Together with decreasing the byproducts formation and boosting precursor supply, the titer of FA reaches 5.09 g/L under fed-batch conditions, indicating a 20-fold improvement compared with the original producing E. coli strain. This work not only establishes a promising microbial platform for industrial level production of FA and its derivatives, but also highlights a convenient and effective strategy to enhance the biosynthesis of chemicals requiring methylation and FADH2-dependent hydroxylation.

Plasma proteomic analysis reveals altered protein abundances in HIV-infected patients with or without non-Hodgkin lymphoma.

The identification of circulating proteins associated with acquired immunodeficiency syndrome-related non-Hodgkin lymphoma (AIDS-NHL) may help in the development of promising biomarkers for screening, diagnosis, treatment, and prognosis. Here, we used quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify differentially expressed proteins (DEPs) in plasma collected from patients with AIDS-NHL and human immunodeficiency virus (HIV)-infected patients without NHL (HIV+ ). Proteins with a log2 (fold change) in abundance >0.26 and p < 0.05 were considered differentially abundant. In total, 84 DEPs were identified, among which 20 were further validated as potential biomarkers, with immunoglobulin and complement components being the most common proteins. Some of the proteins were further verified in a retrospective analysis of the medical records of patients in a larger cohort. These markedly altered proteins were found to mediate pathophysiological pathways that likely contribute to AIDS-NHL pathogenesis, such as the humoral immune response, complement activation, and complement and coagulation cascades. Our findings provide a new molecular understanding of AIDS-NHL pathogenesis and provide new evidence supporting the identification of these proteins as possible biomarkers in AIDS-NHL.

Biosynthesis of allantoin in Escherichia coli via screening a highly effective urate oxidase.

Allantoin is an important fine chemical that can be widely used in pharmaceutical, cosmetic and agricultural industries. Currently, allantoin is mainly produced by plant extraction or chemical synthesis. Due to the cost and environmental concerns, biosynthesis of allantoin from renewable feedstock is much more desirable. However, microbial production of allantoin from simple carbon sources has not yet been achieved so far. In this study, de novo biosynthesis of allantoin was achieved by constructing an artificial biosynthetic pathway. First, screening of efficient urate oxidases and xanthine dehydrogenases enabled allantoin production from hypoxanthine, a natural intermediate in purine metabolic pathway in Escherichia coli. Then, assemble of the entire pathway resulted in 13.9 mg/L allantoin from glucose in shake flask experiments. The titer was further improved to 639.8 mg/L by enhancing the supply of the precursor, redistribution of carbon flux, and reduction of acetate. Finally, scale-up production of allantoin was conducted in a 1-L fermentor under fed-batch culture conditions, which enabled the synthesis of 2360 mg/L allantoin, representing a 170-fold increase compared with the initial strain. This study not only demonstrates the potential for industrial production of allantoin, but also provides a bacterial platform for synthesis of other purines-derived high-value chemicals.

Design and construction of an artificial pathway for biosynthesis of acetaminophen in Escherichia coli.

Acetaminophen (AAP) is one of the most commonly used drug ingredients that possesses antipyretic and analgesic effects. As an unnatural chemical, AAP is commercially produced by chemical processes using petroleum-derived carbohydrates, such as phenol, as raw materials, which is unsustainable and eco-unfriendly. In this study, we report design and construction of an artificial biosynthetic pathway for de novo production of AAP from simple carbon source. By exploring and expanding the substrate repertoire of natural enzymes, we identified and characterized a novel p-aminobenzoic acid (p-ABA) monooxygenase and an p-aminophenol (p-AP) N-acetyltransferase, which enabled the bacterial production of AAP from p-ABA. Then, we constructed an p-ABA over-producer by screening of p-ABA synthases and enhancing glutamine availability, resulting in 836.43 mg/L p-ABA in shake flasks in E. coli. Subsequent assembly of the entire biosynthetic pathway permitted the de novo production of AAP from glycerol for the first time. Finally, pathway engineering by dynamically regulating the expression of pathway genes via a temperature-inducible controller enabled production enhancement of AAP with a titer of 120.03 mg/L. This work not only constructs a microbial platform for AAP production, but also demonstrates design and construction of artificial biosynthetic pathways via discovering novel bioreactions based on existing enzymes.

Multiband Superconductivity with Sign-Preserving Order Parameter in Kagome Superconductor CsV_{3}Sb_{5}.

The superconductivity of a kagome superconductor CsV_{3}Sb_{5} is studied by scanning tunneling microscopy and spectroscopy at ultralow temperature with high resolution. Two kinds of superconducting gaps with multiple sets of coherent peaks and residual zero-energy density of states (DOS) are observed on both half-Cs and Sb surfaces, implying multiband superconductivity. In addition, in-gap states can be induced by magnetic impurities but not by nonmagnetic impurities, suggesting a sign-preserving or s-wave superconducting order parameter. Moreover, the interplay between charge density waves (CDW) and superconductivity differs on various bands, resulting in different density-of-states distributions. Our results suggest that the superconducting gap is likely isotropic on the sections of Fermi surface that play little roles in CDW, and the superconducting gaps on the sections of Fermi surface with anisotropic CDW gaps are likely anisotropic as well. The residual spectral weights at zero energy are attributed to the extremely small superconducting gap on the tiny oval Fermi pockets. Our study provides critical clues for further understanding the superconductivity and its relation to CDW in CsV_{3}Sb_{5}.

Observation of Distinct Spatial Distributions of the Zero and Nonzero Energy Vortex Modes in (Li_{0.84}Fe_{0.16})OHFeSe.

The energy and spatial distributions of vortex bound state in superconductors carry important information about superconducting pairing and the electronic structure. Although discrete vortex states, and sometimes a zero energy mode, had been observed in several iron-based superconductors, their spatial properties are rarely explored. In this study, we used low-temperature scanning tunneling microscopy to measure the vortex state of (Li,Fe)OHFeSe with high spatial resolution. We found that the nonzero energy states display clear spatial oscillations with a period corresponding to bulk Fermi wavelength; while in contrast, the zero energy mode does not show such oscillation, which suggests its distinct electronic origin. Furthermore, the oscillations of positive and negative energy states near E_{F} are found to be clearly out of phase. Based on a two-band model calculation, we show that our observation is more consistent with an s_{++} wave pairing in the bulk of (Li, Fe)OHFeSe, and superconducting topological states on the surface.

Extending the shikimate pathway for microbial production of maleate from glycerol in engineered Escherichia coli.

Maleate is one of the most important unsaturated four-carbon dicarboxylic acids. It serves as an attractive building block in cosmetic, polymer, and pharmaceutical industries. Currently, industrial production of maleate relies mainly on chemical synthesis using benzene or butane as the starting materials under high temperature, which suffers from strict reaction conditions and low product yield. Here, we propose a novel biosynthetic pathway for maleate production in engineered Escherichia coli. We screened a superior salicylate 5-hydroxylase that can catalyze hydroxylation of salicylate into gentisate with high conversion rate. Then, introduction of salicylate biosynthetic pathway and gentisate ring cleavage pathway allowed the synthesis of maleate from glycerol. Further optimizations including enhancement of precursors supply, disruption of competing pathways, and construction of a pyruvate recycling system, boosted maleate titer to 2.4 ± 0.1 g/L in shake flask experiments. Subsequent scale-up biosynthesis of maleate in a 3-L bioreactor under fed-batch culture conditions enabled the production of 14.5 g/L of maleate, indicating a 268-fold improvement compared with the titer generated by the wildtype E. coli strain carrying the entire maleate biosynthetic pathway. This study provided a promising microbial platform for industrial level synthesis of maleate, and demonstrated the highest titer of maleate production in microorganisms so far.

Rewiring the microbial metabolic network for efficient utilization of mixed carbon sources.

Carbon sources represent the most dominant cost factor in the industrial biomanufacturing of products. Thus, it has attracted much attention to seek cheap and renewable feedstocks, such as lignocellulose, crude glycerol, methanol, and carbon dioxide, for biosynthesis of value-added compounds. Co-utilization of these carbon sources by microorganisms not only can reduce the production cost but also serves as a promising approach to improve the carbon yield. However, co-utilization of mixed carbon sources usually suffers from a low utilization rate. In the past few years, the development of metabolic engineering strategies to enhance carbon source co-utilization efficiency by inactivation of carbon catabolite repression has made significant progress. In this article, we provide informative and comprehensive insights into the co-utilization of two or more carbon sources including glucose, xylose, arabinose, glycerol, and C1 compounds, and we put our focus on parallel utilization, synergetic utilization, and complementary utilization of different carbon sources. Our goal is not only to summarize strategies of co-utilization of carbon sources, but also to discuss how to improve the carbon yield and the titer of target products.

Observation of Discrete Conventional Caroli-de Gennes-Matricon States in the Vortex Core of Single-Layer FeSe/SrTiO_{3}.

Using low-temperature scanning tunneling microscopy (STM), we studied the vortex states of single-layer FeSe film on a SrTiO_{3} (100) substrate, and the local behaviors of superconductivity at sample boundaries. We clearly observed multiple discrete Caroli-de Gennes-Matricon states in the vortex core, and quantitative analysis shows their energies well follow the formula: E=µΔ^{2}/E_{F}, where µ is a half integer (±1/2,±3/2,±5/2…) and Δ is the mean superconducting gap over the Fermi surface. Meanwhile, a fully gapped spectrum without states near zero bias is observed at the [110]_{Fe} oriented boundary of 1 and 2 ML FeSe films, and atomic step edge of 1 ML FeSe. Accompanied with theoretical calculations, our results indicate an s-wave pairing without sign change in the high-T_{C} FeSe/SrTiO_{3} superconductor.

Engineering probiotics as living diagnostics and therapeutics for improving human health.

The gut microbiota that inhabit our gastrointestinal tract are well known to play an important role in maintaining human health in many aspects, including facilitating the digestion and absorption of nutrients, protecting against pathogens and regulating immune system. Gut microbiota dysbiosis is associated with a lot of diseases, such as inflammatory bowel disease, allergy, obesity, cardiovascular and neurodegenerative diseases and cancers. With the increasing knowledge of the microbiome, utilization of probiotic bacteria in modulating gut microbiota to prevent and treat a large number of disorders and diseases has gained much interest. In recent years, aided by the continuous development of tools and techniques, engineering probiotic microbes with desired characteristics and functionalities to benefit human health has made significant progress. In this paper, we summarize the recent advances in design and construction of probiotics as living diagnostics and therapeutics for probing and treating a series of diseases including metabolic disorders, inflammation and pathogenic bacteria infections. We also discuss the current challenges and future perspectives in expanding the application of probiotics for disease treatment and detection. We intend to provide insights and ideas for engineering of probiotics to better serve disease therapy and human health.