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The biosynthetic investigations of microbial natural products continuously provide powerful biocatalysts for the preparation of valuable chemicals. Practical methods for preparing (S)-3-aminopiperidine-2,6-dione (2), the pharmacophore of thalidomide (1) and its analog drugs, are highly desired. To develop a biocatalyst for producing (S)-2, we dissected the domain functions of IdgS, which is responsible for the biosynthesis of indigoidine (3), a microbial blue pigment that consists of two 2-like moieties. Our data supported that the L-glutamine tethered to the indigoidine assembly line is first offloaded and cyclized by the thioesterase domain to form (S)-2, which is then dehydrogenated by the oxidation (Ox) domain and finally dimerized to yield 3. Based on this, we developed an IdgS-derived enzyme biocatalyst, IdgS-Ox* R539A, for preparing enantiomerically pure (S)-2. As a proof of concept, one-pot chemoenzymatic synthesis of 1 was achieved by combining the biocatalytic and chemical approaches.

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.

Marine algae and bacteria produce approximately eight billion tonnes of the organosulfur molecule dimethylsulfoniopropionate (DMSP) in Earth's surface oceans annually. DMSP is an antistress compound and, once released into the environment, a major nutrient, signaling molecule, and source of climate-active gases. The methionine transamination pathway for DMSP synthesis is used by most known DMSP-producing algae and bacteria. The S-directed S-adenosylmethionine (SAM)-dependent 4-methylthio-2-hydroxybutyrate (MTHB) S-methyltransferase, encoded by the dsyB/DSYB gene, is the key enzyme of this pathway, generating S-adenosylhomocysteine (SAH) and 4-dimethylsulfonio-2-hydroxybutyrate (DMSHB). DsyB/DSYB, present in most haptophyte and dinoflagellate algae with the highest known intracellular DMSP concentrations, is shown to be far more abundant and transcribed in marine environments than any other known S-methyltransferase gene in DMSP synthesis pathways. Furthermore, we demonstrate in vitro activity of the bacterial DsyB enzyme from Nisaea denitrificans and provide its crystal structure in complex with SAM and SAH-MTHB, which together provide the first important mechanistic insights into a DMSP synthesis enzyme. Structural and mutational analyses imply that DsyB adopts a proximity and desolvation mechanism for the methyl transfer reaction. Sequence analysis suggests that this mechanism may be common to all bacterial DsyB enzymes and also, importantly, eukaryotic DSYB enzymes from e.g., algae that are the major DMSP producers in Earth's surface oceans.

Glucose and xylose are two major components of lignocellulose. Simultaneous consumption of glucose and xylose is critical for engineered microorganisms to produce fuels and chemicals from lignocellulosic biomass. Although many production limitations have been resolved, glucose-induced inhibition of xylose transport remains a challenge. In this study, a cell growth-based screening strategy was designed to identify xylose transporters uninhibited by glucose. The glucose pathway was genetically blocked in Escherichia coli so that glucose functions only as an inhibitor and cells need xylose as the carbon source for survival. Through adaptive evolution, omics analysis and reverse metabolic engineering, a new phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) galactitol transporter (GalABC, encoded by EcolC_1640, EcolC_1641, and EcolC_1642 genes) that is not inhibited by glucose was identified. Inactivation of adenylate cyclase led to increased expression of the EcolC_1642 gene, and a point mutation in gene EcolC_1642 (N13S) further enhanced xylose transport. During the second round of gene mining, AraE and a new ABC transporter (AraFGH) of xylose were identified. A point mutation in the transcription regulator araC (L156I) caused increased expression of araE and araFGH genes without arabinose induction, and a point mutation in araE (D223Y) further enhanced xylose transport. These newly identified xylose transporters can support the simultaneous consumption of glucose and xylose and have potential use in producing chemicals from lignocellulose.

The applications of marker gene concatenation have been advanced to resolve the key questions in the Tree of Life. However, the interphylum evolutionary relationship between Korarchaeota of TACK (Thaumarchaeota, Aigarchaeota, Crenarchaeota, Korarchaeota) and Asgard archaea remains uncertain. We applied a marker gene ranking procedure to examine their evolutionary history. Our updated trees showed confident placements of (1) Korarchaeota as the basal branch to other TACK archaea and as a sister group to Asgard archaea; (2) Njordarchaeota at basal branch to Korarchaeota instead of within Asgard archaea. They highlight the importance of evaluating marker genes for phylogeny inference of the Archaea domain.

Gut microbiota composition is suggested to associate with coronavirus disease 2019 (COVID-19) severity, but the impact of gut microbiota on health outcomes is largely unclear. We recruited 81 individuals from Wuhan, China, including 13 asymptomatic infection cases (Group A), 24 COVID-19 convalescents with adverse outcomes (Group C), 31 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) re-positive cases (Group D), and 13 non-COVID-19 healthy controls (Group H). The microbial features of Groups A and D were similar and exhibited higher gut microbial diversity and more abundant short-chain fatty acid (SCFA)-producing species than Group C. Group C was enriched with opportunistic pathogens and virulence factors related to adhesion and toxin production. The abundance of SCFA-producing species was negatively correlated, while Escherichia coli was positively correlated with adverse outcomes. All three groups (A, C, and D) were enriched with the mucus-degrading species Akkermansia muciniphila, but decreased with Bacteroides-encoded carbohydrate-active enzymes. The pathways of vitamin B6 metabolic and folate biosynthesis were decreased, while selenocompound metabolism was increased in the three groups. Specifically, the secondary bile acid (BA) metabolic pathway was enriched in Group A. Antibiotic resistance genes were common among the three groups. Conclusively, the gut microbiota was related to the health outcomes of COVID-19. Dietary supplementations (SCFAs, BA, selenium, folate, vitamin B6) may be beneficial to COVID-19 patients.

Nonhuman primates (NHPs) such as monkeys are the closest living relatives to humans and are the best available models for causative studies of human health and diseases. Gut microbiomes are intensively involved in host health. In this study, by large-scale cultivation of microbes from fecal samples of monkeys, we obtained previously uncultured bacterial species and constructed a Macaca fascicularis Gut Microbial Biobank (MfGMB). The MfGMB consisted of 250 strains that represent 97 species of 63 genera, 25 families, and 4 phyla. The information of the 250 strains and the genomes of 97 cultured species are publicly accessible. The MfGMB represented nearly 50% of core gut microbial compositions at the genus level and covered over 80% of the KO-based known gut microbiome functions of M. fascicularis. Data mining showed that the bacterial species in the MfGMB were prevalent not only in NHPs gut microbiomes but also in human gut microbiomes. This study will help the understanding and future investigations on how gut microbiomes interact with their mammalian hosts.

Biological denitrification is a crucial process in the nitrogen biogeochemical cycle, and Thermus has been reported to be a significant heterotrophic denitrifier in terrestrial geothermal environments. However, neither the denitrification potential nor the evolutionary history of denitrification genes in the genus Thermus or phylum Deinococcota is well understood. Here, we performed a comparative analysis of 23 Thermus genomes and identified denitrification genes in 15 Thermus strains. We confirmed that Thermus harbors an incomplete denitrification pathway as none of the strains contain the nosZ gene. Ancestral character state reconstructions and phylogenetic analyses showed that narG, nirS, and norB genes were acquired by the last common ancestor of Thermales and were inherited vertically. In contrast, nirK of Thermales was acquired via two distinct horizontal gene transfers from Proteobacteria to the genus Caldithermus and from an unknown donor to the common ancestor of all known Thermus species except Thermus filiformis. This study expands our understanding of the genomic potential for incomplete denitrification in Thermus, revealing a largely vertical evolutionary history of the denitrification pathway in the Thermaceae, and supporting the important role for Thermus as an important heterotrophic denitrifier in geothermal environments.

Antimicrobial susceptibility tests (ASTs) are pivotal in combating multidrug resistant pathogens, yet they can be time-consuming, labor-intensive, and unstable. Using the AST of tigecycline for sepsis as the main model, here we establish an automated system of Clinical Antimicrobials Susceptibility Test Ramanometry (CAST-R), based on D₂O-probed Raman microspectroscopy. Featuring a liquid robot for sample pretreatment and a machine learning-based control scheme for data acquisition and quality control, the 3-h, automated CAST-R process accelerates AST by >10-fold, processes 96 paralleled antibiotic-exposure reactions, and produces high-quality Raman spectra. The Expedited Minimal Inhibitory Concentration via Metabolic Activity is proposed as a quantitative and broadly applicable parameter for metabolism-based AST, which shows 99% essential agreement and 93% categorical agreement with the broth microdilution method (BMD) when tested on 100 Acinetobacter baumannii isolates. Further tests on 26 clinically positive blood samples for eight antimicrobials, including tigecycline, meropenem, ceftazidime, ampicillin/sulbactam, oxacillin, clindamycin, vancomycin, and levofloxacin reveal 93% categorical agreement with BMD-based results. The automation, speed, reliability, and general applicability of CAST-R suggest its potential utility for guiding the clinical administration of antimicrobials.