Species-specific design of artificial promoters by transfer-learning based generative deep-learning model.

Native prokaryotic promoters share common sequence patterns, but are species dependent. For understudied species with limited data, it is challenging to predict the strength of existing promoters and generate novel promoters. Here, we developed PromoGen, a collection of nucleotide language models to generate species-specific functional promoters, across dozens of species in a data and parameter efficient way. Twenty-seven species-specific models in this collection were finetuned from the pretrained model which was trained on multi-species promoters. When systematically compared with native promoters, the Escherichia coli- and Bacillus subtilis-specific artificial PromoGen-generated promoters (PGPs) were demonstrated to hold all distribution patterns of native promoters. A regression model was developed to score generated either by PromoGen or by another competitive neural network, and the overall score of PGPs is higher. Encouraged by in silico analysis, we further experimentally characterized twenty-two B. subtilis PGPs, results showed that four of tested PGPs reached the strong promoter level while all were active. Furthermore, we developed a user-friendly website to generate species-specific promoters for 27 different species by PromoGen. This work presented an efficient deep-learning strategy for de novo species-specific promoter generation even with limited datasets, providing valuable promoter toolboxes especially for the metabolic engineering of understudied microorganisms.

Engineering amino acid residues of pentacyclic triterpene synthases for improving the activity.

Pentacyclic triterpenoids exhibit a wide range of biological activities which have wide applications in the food, cosmetics, and pharmaceutical industries. High-performance chassis strains have been developed for the production of various pentacyclic triterpenoids, e.g., lupane-type and oleanane-type triterpenoids. The production of common pentacyclic triterpenes and their derivatives is limited by the poor activity of typical pentacyclic triterpene synthases (PTSs). However, a general strategy applicable to typical PTSs is still lacking. As typical pentacyclic triterpenes are derived from the baccharenyl cation, engineering the non-active-site residues in the MXXXXR motif might be beneficial for the catalytic efficiencies of typical PTSs by the stabilization of the baccharenyl cation. Here, we develop a general strategy for improving the activity of typical PTSs. As a proof of concept, the activity of three PTSs such as lupeol synthase, ß-amyrin synthase, and α-amyrin synthases was significantly increased up to 7.3-fold by site-directed saturation mutagenesis. This strategy could be applied to improve the activity of various typical PTSs. KEY POINTS: • The strategy could be applied to typical PTSs for improving the activity. • The catalytic activity of typical PTSs was significantly increased.

Development of meso-Five-Membered Heterocycle BODIPY-Based AIE Fluorescent Probes for Dual-Organelle Viscosity Imaging.

Fluorescent rotors with aggregation-induced emission (AIE) and organelle-targeting properties have attracted great attention for sensing subcellular viscosity changes, which could help understand the relationships of abnormal fluctuations with many associated diseases. Despite the numerous efforts spent, it remains rare and urgent to explore the dual-organelle targeting probes and their structural relationships with viscosity-responsive and AIE properties. Therefore, in this work, we reported four meso-five-membered heterocycle-substituted BODIPY-based fluorescent probes, explored their viscosity-responsive and AIE properties, and further investigated their subcellular localization and viscosity-sensing applications in living cells. Interestingly, the meso-thiazole probe 1 showed both good viscosity-responsive and AIE (in pure water) properties and could successfully target both mitochondria and lysosomes, further imaging cellular viscosity changes by treating lipopolysaccharide and nystatin, attributing to the free rotation and potential dual-organelle targeting ability of the meso-thiazole group. The meso-benzothiophene probe 3 with a saturated sulfur only showed good viscosity-responsive properties in living cells with the aggregation-caused quenching effect and no subcellular localization. The meso-imidazole probe 2 showed the AIE phenomenon without an obvious viscosity-responsive property with a C═N bond, while the meso-benzopyrrole probe 4 displayed fluorescence quenching in polar solvents. Therefore, for the first time, we investigated the structure-property relationships of four meso-five-membered heterocycle-substituted BODIPY-based fluorescent rotors with viscosity-responsive and AIE properties, and among these, 1 with a C═N bond and a saturated sulfur on the meso-thiazole, potentially contributing to their corresponding AIE and viscosity-responsive properties, served as a sensitive AIE fluorescent rotor for imaging dual-organelle viscosity in both mitochondria and lysosomes.

M1 polarization enhances the antitumor activity of chimeric antigen receptor macrophages in solid tumors.

BACKGROUND: Chimeric antigen receptor macrophage (CAR-M) therapy is a novel cancer immunotherapy approach that integrates CAR structure and macrophage functions. CAR-M therapy has shown unique and impressive antitumor effects in immunotherapy for solid tumors. However, the polarization state of macrophages can affect the antitumor effect of CAR-M. We hypothesized that the antitumor activity of CAR-Ms may be further improved after inducing M1-type polarization. METHODS: In this report, we constructed a novel HER2-targeting CAR-M, which was composed of humanized anti-HER2 scFv, CD28 hinge region and FcγRI transmembrane domain and intracellular domain. Phagocytosis, tumor-killing capacities, and cytokine release of CAR-Ms were detected with or without M1-polarization pretreatment. Several syngeneic tumor models were used to monitor the in vivo antitumor activity of M1-polarized CAR-Ms. RESULTS: After polarization with LPS combined with interferon-γ in vitro, we found that the phagocytic and tumor-killing capacities of CAR-Ms against target cells were significantly enhanced. The expression of costimulatory molecules and proinflammatory cytokines was also significantly increased after polarization. By establishing several syngeneic tumor models in vivo, we also demonstrated that infusing polarized M1-type CAR-Ms could effectively suppress tumor progression and prolong the survival of tumor-bearing mice with enhanced cytotoxicity. CONCLUSIONS: We demonstrated that our novel CAR-M can effectively eliminate HER2-positive tumor cells both in vitro and in vivo, and M1 polarization significantly enhanced the antitumor ability of CAR-M, resulting in a stronger therapeutic effect in solid cancer immunotherapy.

A tRNAModification-based strategy for Identifying amiNo acid Overproducers (AMINO).

Amino acids have a multi-billion-dollar market with rising demand, prompting the development of high-performance microbial factories. However, a general screening strategy applicable to all proteinogenic and non-proteinogenic amino acids is still lacking. Modification of the critical structure of tRNA could decrease the aminoacylation level of tRNA catalyzed by aminoacyl-tRNA synthetases. Involved in a two-substrate sequential reaction, amino acids with increased concentration could elevate the reduced aminoacylation rate caused by specific tRNA modification. Here, we developed a selection system for overproducers of specific amino acids using corresponding engineered tRNAs and marker genes. As a proof-of-concept, overproducers of five amino acids such as L-tryptophan were screened out by growth-based and/or fluorescence-activated cell sorting (FACS)-based screening from random mutation libraries of Escherichia coli and Corynebacterium glutamicum, respectively. This study provided a universal strategy that could be applied to screen overproducers of proteinogenic and non-proteinogenic amino acids in amber-stop-codon-recoded or non-recoded hosts.

Strategies for improving the production of bio-based vanillin.

Vanillin (4-hydroxy-3-methoxybenzaldehyde) is one of the most popular flavors with wide applications in food, fragrance, and pharmaceutical industries. However, the high cost and limited yield of plant extraction failed to meet the vast market demand of natural vanillin. Vanillin biotechnology has emerged as a sustainable and cost-effective alternative to supply vanillin. In this review, we explored recent advances in vanillin biosynthesis and highlighted the potential of vanillin biotechnology. In particular, we addressed key challenges in using microorganisms and provided promising approaches for improving vanillin production with a special focus on chassis development, pathway construction and process optimization. Future directions of vanillin biosynthesis using inexpensive precursors are also thoroughly discussed.

Accuracy and eligibility of Bonwill⁃Hawley arch form established by CBCT image for dental crowding measurement: a comparative study with the conventional brass wire and caliper methods.

OBJECTIVES: The purpose of this study was to develop a novel Bonwill⁃Hawley method (Bonwill⁃Hawley arch form based on CBCT image) for the assessment of dental crowding, and to investigate and compare the accuracy and eligibility with the conventional brass wire and caliper methods under different crowding conditions. MATERIAL AND METHODS: Sixty patients with the pair of plaster casts and CBCT data were collected. All the casts were marked and transformed into digital models using iTero scanner, and imported into OrthoCAD software to measure the required space. Using the conventional brass wire (M1) and caliper methods (M2), the available space and dental crowding were measured and calculated basing on digital models, respectively. Correspondingly, the axial planes in the level of dental arches were oriented and captured from the CBCT images to draw the Bonwill⁃Hawley arch forms (M3), which were used to measure and calculate the available space and dental crowding. For each method, intra and inter-examiner reliabilities were evaluated with intra-class correlation coefficients (ICCs). Wilcoxon test and Kruskal-Wallis test were performed for statistically analyzing the discrepancy among different groups. RESULTS: Both intra- and inter-examiner reliability were generally excellent for all parameters obtained by the three methods, except for the dental crowding measured using M1(ICC: 0.473/0.261). The dental crowding measured using M2 were significantly increased in mild, moderate and severe-crowding groups compared with M1. However, no significant difference was detected between M1 and M3 in severe-crowding group (maxilla, p = 0.108 > 0.05; mandible, p = 0.074 > 0.05). With the deterioration of crowding condition, the discrepancy of dental crowding between M1 and M2, or M1 and M3 were significantly decreased (maxilla, M2-M1, mild VS serve, p = 0.003 < 0.05; maxilla, M3-M1, mild VS serve, p = 0.003 < 0.05; mandible, M2-M1, mild VS serve, p = 0.000 < 0.001; mandible, M3-M1, mild VS serve, p = 0.043 < 0.05). CONCLUSION: Dental crowding measured using the novel Bonwill⁃Hawley method was relatively greater than the caliper method, but not exceeding the brass wire method, which wound gradually come close to the brass wire method with the deterioration of crowding condition. CLINICAL RELEVANCE: The Bonwill⁃Hawley method basing on CBCT image proved to be a reliable and acceptable choice for orthodontists to analyze the dental crowding.

Polyphenol Utilization Proteins in the Human Gut Microbiome.

Dietary polyphenols can significantly benefit human health, but their bioavailability is metabolically controlled by human gut microbiota. To facilitate the study of polyphenol metabolism for human gut health, we have manually curated experimentally characterized polyphenol utilization proteins (PUPs) from published literature. This resulted in 60 experimentally characterized PUPs (named seeds) with various metadata, such as species and substrate. Further database search found 107,851 homologs of the seeds from UniProt and UHGP (unified human gastrointestinal protein) databases. All PUP seeds and homologs were classified into protein classes, families, and subfamilies based on Enzyme Commission (EC) numbers, Pfam (protein family) domains, and sequence similarity networks. By locating PUP homologs in the genomes of UHGP, we have identified 1,074 physically linked PUP gene clusters (PGCs), which are potentially involved in polyphenol metabolism in the human gut. The gut microbiome of Africans was consistently ranked the top in terms of the abundance and prevalence of PUP homologs and PGCs among all geographical continents. This reflects the fact that dietary polyphenols are consumed by the African population more commonly than by other populations, such as Europeans and North Americans. A case study of the Hadza hunter-gatherer microbiome verified the feasibility of using dbPUP to profile metagenomic data for biologically meaningful discovery, suggesting an association between diet and PUP abundance. A Pfam domain enrichment analysis of PGCs identified a number of putatively novel PUP families. Lastly, a user-friendly web interface (https://bcb.unl.edu/dbpup/) provides all the data online to facilitate the research of polyphenol metabolism for improved human health. IMPORTANCE Long-term consumption of polyphenol-rich foods has been shown to lower the risk of various human diseases, such as cardiovascular diseases, cancers, and metabolic diseases. Raw polyphenols are often enzymatically processed by gut microbiome, which contains various polyphenol utilization proteins (PUPs) to produce metabolites with much higher bioaccessibility to gastrointestinal cells. This study delivered dbPUP as an online database for experimentally characterized PUPs and their homologs in human gut microbiome. This work also performed a systematic classification of PUPs into enzyme classes, families, and subfamilies. The signature Pfam domains were identified for PUP families, enabling conserved domain-based PUP annotation. This standardized sequence similarity-based PUP classification system offered a guideline for the future inclusion of new experimentally characterized PUPs and the creation of new PUP families. An in-depth data analysis was further conducted on PUP homologs and physically linked PUP gene clusters (PGCs) in gut microbiomes of different human populations.

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.

Investigating a Boronate-Affinity-Guided Acylation Reaction for Labelling Native Antibodies.

The excellent molecular recognition capabilities of monoclonal antibodies (mAbs) have opened up exciting opportunities for biotherapeutic discovery. Taking advantage of the full potential of this tool necessitates affinity ligands capable of conjugating directly with small molecules to a defined degree of biorthogonality, especially when modifying natural Abs. Herein, a bioorthogonal boronate-affinity-based Ab ligand featuring a 4-(dimethylamino)pyridine and an S-aryl thioester to label full-length Abs is reported. The photoactivatable linker in the acyl donor facilitated purification of azide-labelled Ab (N3 -Ab) was quantitatively cleaved upon brief exposure to UV light while retaining the original Ab activity. Click reactions enabled the precise addition of biotin, a fluorophore, and a pharmacological agent to the purified N3 -Abs. The resulting immunoconjugate showed selectivity against targeted cells. Bioorthogonal traceless design and reagentless purification allow this strategy to be a powerful tool to engineer native antibodies amenable to therapeutic intervention.

Efficient Large-Scale and Scarless Genome Engineering Enables the Construction and Screening of Bacillus subtilis Biofuel Overproducers.

Bacillus subtilis is a versatile microbial cell factory that can produce valuable proteins and value-added chemicals. Long fragment editing techniques are of great importance for accelerating bacterial genome engineering to obtain desirable and genetically stable host strains. Herein, we develop an efficient CRISPR-Cas9 method for large-scale and scarless genome engineering in the Bacillus subtilis genome, which can delete up to 134.3 kb DNA fragments, 3.5 times as long as the previous report, with a positivity rate of 100%. The effects of using a heterologous NHEJ system, linear donor DNA, and various donor DNA length on the engineering efficiencies were also investigated. The CRISPR-Cas9 method was then utilized for Bacillus subtilis genome simplification and construction of a series of individual and cumulative deletion mutants, which are further screened for overproducer of isobutanol, a new generation biofuel. These results suggest that the method is a powerful genome engineering tool for constructing and screening engineered host strains with enhanced capabilities, highlighting the potential for synthetic biology and metabolic engineering.

Blinatumomab-induced T cell activation at single cell transcriptome resolution.

BACKGROUND: Bi-specific T-cell engager (BiTE) antibody is a class of bispecific antibodies designed for cancer immunotherapy. Blinatumomab is the first approved BiTE to treat acute B cell lymphoblastic leukemia (B-ALL). It brings killer T and target B cells into close proximity, activating patient's autologous T cells to kill malignant B cells via mechanisms such as cytolytic immune synapse formation and inflammatory cytokine production. However, the activated T-cell subtypes and the target cell-dependent T cell responses induced by blinatumomab, as well as the mechanisms of resistance to blinatumomab therapy are largely unknown. RESULTS: In this study, we performed single-cell sequencing analysis to identify transcriptional changes in T cells following blinatumomab-induced T cell activation using single cells from both, a human cell line model and a patient-derived model of blinatumomab-mediated cytotoxicity. In total, the transcriptome of 17,920 single T cells from the cell line model and 2271 single T cells from patient samples were analyzed. We found that CD8+ effector memory T cells, CD4+ central memory T cells, naïve T cells, and regulatory T cells were activated after blinatumomab treatment. Here, blinatumomab-induced transcriptional changes reflected the functional immune activity of the blinatumomab-activated T cells, including the upregulation of pathways such as the immune system, glycolysis, IFNA signaling, gap junctions, and IFNG signaling. Co-stimulatory (TNFRSF4 and TNFRSF18) and co-inhibitory (LAG3) receptors were similarly upregulated in blinatumomab-activated T cells, indicating ligand-dependent T cell functions. Particularly, B-ALL cell expression of TNFSF4, which encodes the ligand of T cell co-stimulatory receptor TNFRSF4, was found positively correlated with the response to blinatumomab treatment. Furthermore, recombinant human TNFSF4 protein enhanced the cytotoxic activity of blinatumomab against B-ALL cells. CONCLUSION: These results reveal a target cell-dependent mechanism of T-cell activation by blinatumomab and suggest that TNFSF4 may be responsible for the resistant mechanism and a potential target for combination therapy with blinatumomab, to treat B-ALL or other B-cell malignancies.

Cellulosimicrobium composti sp. nov., a thermophilic bacterium isolated from compost.

A Gram-stain-positive, yellow-pigmented, non-motile actinobacterial strain, designated as BIT-GX5T, was isolated from a sesame husks compost collected in Beijing, PR China. This bacterium was found to be able to grow in the temperature range from 16 to 50 °C and had an optimal growth temperature at 45 °C. Its taxonomic position was analysed using a polyphasic approach. The 16S rRNA gene sequence (1482 bp) of strain BIT-GX5T was most similar to Cellulosimicrobium funkei ATCC BAA-886T (99.45%), Cellulosimicrobium cellulans LMG 16121T (99.17%) and Cellulosimicrobium marinum RS-7-4T (98.75%). The results of phylogenetic analyses, based on the 16S rRNA gene, concatenated sequences of five housekeeping genes (gyrB, rpoB, recA, atpD and trpB) and genome sequences, placed strain BIT-GX5T in a separate lineage among the genus Cellulosimicrobium within the family Promicromonosporaceae. The major polar lipids of strain BIT-GX5T were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, aminophospholipid and aminolipid. The major isoprenoid quinone was MK-9(H4), while the cell-wall sugars were galactose, rhamnose, glucose and mannose. The peptidoglycan type was A4α l-Lys-d-Ser-d-Asp. The major fatty acids were anteiso-C15:0 and iso-C15: 0, which were similar to other members in the genus Cellulosimicrobium. Results of in silico DNA-DNA hybridization and average nucleotide identity calculations plus physiological and biochemical tests exhibited the genotypic and phenotypic differentiation of strain BIT-GX5T from the other members of the genus Cellulosimicrobium. Therefore, strain BIT-GX5T is considered to represent a novel species within the genus Cellulosimicrobium, for which the name Cellulosimicrobium composti sp. nov. is proposed. The type strain is BIT-GX5T (= CGMCC 1.17687T = KCTC 49391T).

Metabolic engineering of Corynebacterium glutamicum for producing branched chain amino acids.

BACKGROUND: Branched chain amino acids (BCAAs) are widely applied in the food, pharmaceutical, and animal feed industries. Traditional chemical synthetic and enzymatic BCAAs production in vitro has been hampered by expensive raw materials, harsh reaction conditions, and environmental pollution. Microbial metabolic engineering has attracted considerable attention as an alternative method for BCAAs biosynthesis because it is environmentally friendly and delivers high yield. MAIN TEXT: Corynebacterium glutamicum (C. glutamicum) possesses clear genetic background and mature gene manipulation toolbox, and has been utilized as industrial host for producing BCAAs. Acetohydroxy acid synthase (AHAS) is a crucial enzyme in the BCAAs biosynthetic pathway of C. glutamicum, but feedback inhibition is a disadvantage. We therefore reviewed AHAS modifications that relieve feedback inhibition and then investigated the importance of AHAS modifications in regulating production ratios of three BCAAs. We have comprehensively summarized and discussed metabolic engineering strategies to promote BCAAs synthesis in C. glutamicum and offer solutions to the barriers associated with BCAAs biosynthesis. We also considered the future applications of strains that could produce abundant amounts of BCAAs. CONCLUSIONS: Branched chain amino acids have been synthesized by engineering the metabolism of C. glutamicum. Future investigations should focus on the feedback inhibition and/or transcription attenuation mechanisms of crucial enzymes. Enzymes with substrate specificity should be developed and applied to the production of individual BCAAs. The strategies used to construct strains producing BCAAs provide guidance for the biosynthesis of other high value-added compounds.

Intestinirhabdus alba gen. nov., sp. nov., a novel genus of the family Enterobacteriaceae, isolated from the gut of plastic-eating larvae of the Coleoptera insect Zophobas atratus.

A bacterial strain, BIT-B35T, was isolated from the gut of plastic-eating larvae of the Coleoptera insect Zophobas atratus. Its taxonomic position was determined by using a polyphasic approach. Cells were white-pigmented, Gram-stain-negative, motile short rods with terminal flagella. The 16S rRNA gene sequence (1411 bp) of strain BIT-B35T showed highest similarity (98.1%) to Escherichia fergusonii ATCC 35469T and Citrobacter koseri LMG 5519T. The results of phylogenetic analyses, based on the 16S rRNA gene, concatenated sequences of seven housekeeping genes (atpD, gyrB, infB, rpoB, pyrG, fusA and leuS) and genome sequences, placed strain BIT-B35T in a separate lineage among the family of Enterobacteriaceae. The major fatty acids were C16 : 0, C17 : 0 cyclo and C19 : 0 cyclo ω8c. The genomic DNA G+C content of strain BIT-B35T was 57.1 mol%. The chemotaxonomic data plus results of physiological and biochemical tests also distinguished strain BIT-B35T from members of other genera within the family Enterobacteriaceae. Therefore, strain BIT-B35T is considered to represent a novel species of a novel genus within the family Enterobacteriaceae, for which the name Intestinirhabdus alba gen. nov., sp. nov. is proposed. The type strain is BIT-B35T (=CGMCC 1.17042T=KCTC 72448T).

Myroides albus sp. nov., isolated from the gut of plastic-eating larvae of the coleopteran insect Zophobas atratus.

A bacterial strain, BIT-d1T, was isolated from the gut of plastic-eating larvae of the coleopteran insect Zophobas atratus. Its taxonomic position was analysed using a polyphasic approach. Cells were white-pigmented, Gram-stain-negative, non-motile, long rods without flagella. The 16S rRNA gene sequence (1401 bp) of strain BIT-d1T showed highest similarity (98.0%) to Myroides pelagicus SM1T and 96.6~92.6 % similarity to the other species of the genus Myroides. The results of phylogenetic analyses, based on the 16S rRNA gene, concatenated sequences of six housekeeping genes (gyrB, dnaK, tuf, murG, atpA and glyA) and genome sequences, placed strain BIT-d1T in a separate lineage among the genus Myroides, family Flavobacteriaceae. The major isoprenoid quinone was menaquinone-6 (MK-6) and the major fatty acids were C15 : 0 iso, C17 : 0 iso 3-OH and summed feature 9 (comprising iso-C17 : 1 ω9c and/or C16 : 0 10-methyl), which were similar to other members in the genus Myroides. In silico DNA-DNA hybridization and average nucleotide identity calculations plus physiological and biochemical tests exhibited the genotypic and phenotypic differentiation of strain BIT-d1T from the other members of the genus Myroides. Therefore, strain BIT-d1T is considered to represent a novel species within the genus Myroides, for which the name Myroides albus sp. nov is proposed. The type strain is BIT-d1T (=CGMCC 1.17043T=KCTC 72447T).

Mixta tenebrionis sp. nov., isolated from the gut of the plastic-eating mealworm Tenebrio molitor L.

A bacterial strain, BIT-26T, was isolated from the gut of plastic-eating mealworm Tenebrio molitor L. The taxonomic position of this new isolate was investigated by using a polyphasic approach. Cells of the strain were Gram-stain-negative, facultatively anaerobic, motile rods with peritrichous flagella. The 16S rRNA gene sequence (1412 bp) of strain BIT-26T showed the highest similarity (97.4 %) to Erwinia piriflorinigrans CFBP 5888T, followed by Citrobacter sedlakii NBRC 105722T (97.3 %), Mixta calida LMG 25383T (97.3 %), Cronobacter muytjensii ATCC 51329T (97.2 %) and Mixta theicola QC88-366 T (97.2 %). The results of phylogenetic analyses, based on the 16S rRNA gene and concatenated sequences of four housekeeping genes (atpD, gyrB, infB and rpoB), placed strain BIT-26T within the genus Mixta of the family Erwiniaceae. This affiliation was also supported by the chemotaxonomic data. Strain BIT-26T had similar predominant fatty acids, including C12 : 0, C14 : 0, C16 : 0, C17 : 0 cyclo and C19 : 0 cyclo ω8c, to species of the genus Mixta. In silico DNA-DNA hybridization and average nucleotide identity calculations plus physiological and biochemical tests allowed the genotypic and phenotypic differentiation of strain BIT-26T from other species of the genus Mixta with validly published names. Therefore, strain BIT-26T is considered to represent a novel species, for which the name Mixta tenebrionis sp. nov is proposed. The type strain is BIT-26T (=CGMCC 1.17041T=KCTC 72449T).

Genetic engineering of non-native hosts for 1-butanol production and its challenges: a review.

BACKGROUND: Owing to the increase in energy consumption, fossil fuel resources are gradually depleting which has led to the growing environmental concerns; therefore, scientists are being urged to produce sustainable and ecofriendly fuels. Thus, there is a growing interest in the generation of biofuels from renewable energy resources using microbial fermentation. MAIN TEXT: Butanol is a promising biofuel that can substitute for gasoline; unfortunately, natural microorganisms pose challenges for the economical production of 1-butanol at an industrial scale. The availability of genetic and molecular tools to engineer existing native pathways or create synthetic pathways have made non-native hosts a good choice for the production of 1-butanol from renewable resources. Non-native hosts have several distinct advantages, including using of cost-efficient feedstock, solvent tolerant and reduction of contamination risk. Therefore, engineering non-native hosts to produce biofuels is a promising approach towards achieving sustainability. This paper reviews the currently employed strategies and synthetic biology approaches used to produce 1-butanol in non-native hosts over the past few years. In addition, current challenges faced in using non-native hosts and the possible solutions that can help improve 1-butanol production are also discussed. CONCLUSION: Non-native organisms have the potential to realize commercial production of 1- butanol from renewable resources. Future research should focus on substrate utilization, cofactor imbalance, and promoter selection to boost 1-butanol production in non-native hosts. Moreover, the application of robust genetic engineering approaches is required for metabolic engineering of microorganisms to make them industrially feasible for 1-butanol production.

Reversed paired-gRNA plasmid cloning strategy for efficient genome editing in Escherichia coli.

BACKGROUND: Co-expression of two distinct guide RNAs (gRNAs) has been used to facilitate the application of CRISPR/Cas9 system in fields such as large genomic deletion. The paired gRNAs are often placed adjacently in the same direction and expressed individually by two identical promoters, constituting direct repeats (DRs) which are susceptible to self-homologous recombination. As a result, the paired-gRNA plasmids cannot remain stable, which greatly prevents extensible applications of CRISPR/Cas9 system. RESULTS: To address this limitation, different DRs-involved paired-gRNA plasmids were designed and the events of recombination were characterized. Deletion between DRs occurred with high frequencies during plasmid construction and subsequent plasmid propagation. This recombination event was RecA-independent, which agreed with the replication slippage model. To increase plasmid stability, a reversed paired-gRNA plasmids (RPGPs) cloning strategy was developed by converting DRs to the more stable invert repeats (IRs), which completely eliminated DRs-induced recombination. Using RPGPs, rapid deletion of chromosome fragments up to 100 kb with an efficiency of 83.33% was achieved in Escherichia coli. CONCLUSIONS: The RPGPs cloning strategy serves as a general solution to avoid plasmid RecA-independent recombination. It can be adapted to applications that rely on paired gRNAs or repeated genetic parts.

Efficient long fragment editing technique enables large-scale and scarless bacterial genome engineering.

Bacteria are versatile living systems that enhance our understanding of nature and enable biosynthesis of valuable chemicals. Long fragment editing techniques are of great importance for accelerating bacterial genome engineering to obtain desirable and genetically stable strains. However, the existing genome editing methods cannot meet the needs of engineers. We herein report an efficient long fragment editing method for large-scale and scarless genome engineering in Escherichia coli. The method enabled us to insert DNA fragments up to 12 kb into the genome and to delete DNA fragments up to 186.7 kb from the genome, with positive rates over 95%. We applied this method for E. coli genome simplification, resulting in 12 individual deletion mutants and four cumulative deletion mutants. The simplest genome lost a total of 370.6 kb of DNA sequence containing 364 open reading frames. Additionally, we applied this technique to metabolic engineering and obtained a genetically stable plasmid-independent isobutanol production strain that produced 1.3 g/L isobutanol via shake-flask fermentation. These results suggest that the method is a powerful genome engineering tool, highlighting its potential to be applied in synthetic biology and metabolic engineering. KEY POINTS: • This article reports an efficient genome engineering tool for E. coli. • The tool is advantageous for the manipulations of long DNA fragments. • The tool has been successfully applied for genome simplification. • The tool has been successfully applied for metabolic engineering.