A citric acid cycle-deficient Escherichia coli as an efficient chassis for aerobic fermentations.

Tricarboxylic acid cycle (TCA cycle) plays an important role for aerobic growth of heterotrophic bacteria. Theoretically, eliminating TCA cycle would decrease carbon dissipation and facilitate chemicals biosynthesis. Here, we construct an E. coli strain without a functional TCA cycle that can serve as a versatile chassis for chemicals biosynthesis. We first use adaptive laboratory evolution to recover aerobic growth in minimal medium of TCA cycle-deficient E. coli. Inactivation of succinate dehydrogenase is a key event in the evolutionary trajectory. Supply of succinyl-CoA is identified as the growth limiting factor. By replacing endogenous succinyl-CoA dependent enzymes, we obtain an optimized TCA cycle-deficient E. coli strain. As a proof of concept, the strain is engineered for high-yield production of four separate products. This work enhances our understanding of the role of the TCA cycle in E. coli metabolism and demonstrates the advantages of using TCA cycle-deficient E. coli strain for biotechnological applications.

CRISPR-Cas9 assisted non-homologous end joining genome editing system of Halomonas bluephagenesis for large DNA fragment deletion.

BACKGROUND: Halophiles possess several unique properties and have broad biotechnological applications including industrial biotechnology production. Halomonas spp., especially Halomonas bluephagenesis, have been engineered to produce various biopolyesters such as polyhydroxyalkanoates (PHA), some proteins, small molecular compounds, organic acids, and has the potential to become a chassis cell for the next-generation of industrial biotechnology (NGIB) owing to its simple culture, fast growth, contamination-resistant, low production cost, and high production value. An efficient genome editing system is the key for its engineering and application. However, the efficiency of the established CRISPR-Cas-homologous recombination (HR) gene editing tool for large DNA fragments was still relatively low. In this study, we firstly report a CRISPR-Cas9 gene editing system combined with a non-homologous end joining (NHEJ) repair system for efficient large DNA fragment deletion in Halomonas bluephagenesis. RESULTS: Three different NHEJ repair systems were selected and functionally identified in Halomonas bluephagenesis TD01. The NHEJ system from M. tuberculosis H37Rv (Mt-NHEJ) can functionally work in H. bluephagenesis TD01, resulting in base deletion of different lengths for different genes and some random base insertions. Factors affecting knockout efficiencies, such as the number and position of sgRNAs on the DNA double-strands, the Cas9 protein promoter, and the interaction between the HR and the NHEJ repair system, were further investigated. Finally, the optimized CRISPR-Cas9-NHEJ editing system was able to delete DNA fragments up to 50 kb rapidly with high efficiency of 31.3%, when three sgRNAs on the Crick/Watson/Watson DNA double-strands and the arabinose-induced promoter Para for Cas9 were used, along with the background expression of the HR repair system. CONCLUSIONS: This was the first report of CRISPR-Cas9 gene editing system combined with a non-homologous end joining (NHEJ) repair system for efficient large DNA fragment deletion in Halomonas spp. These results not only suggest that this editing system is a powerful genome engineering tool for constructing chassis cells in Halomonas, but also extend the application of the NHEJ repair system.

Construction of an artificial phosphoketolase pathway that efficiently catabolizes multiple carbon sources to acetyl-CoA.

The canonical glycolysis pathway is responsible for converting glucose into 2 molecules of acetyl-coenzyme A (acetyl-CoA) through a cascade of 11 biochemical reactions. Here, we have designed and constructed an artificial phosphoketolase (APK) pathway, which consists of only 3 types of biochemical reactions. The core enzyme in this pathway is phosphoketolase, while phosphatase and isomerase act as auxiliary enzymes. The APK pathway has the potential to achieve a 100% carbon yield to acetyl-CoA from any monosaccharide by integrating a one-carbon condensation reaction. We tested the APK pathway in vitro, demonstrating that it could efficiently catabolize typical C1-C6 carbohydrates to acetyl-CoA with yields ranging from 83% to 95%. Furthermore, we engineered Escherichia coli stain capable of growth utilizing APK pathway when glycerol act as a carbon source. This novel catabolic pathway holds promising route for future biomanufacturing and offering a stoichiometric production platform using multiple carbon sources.

De novo artificial synthesis of hexoses from carbon dioxide.

Developing artificial "CO2-sugar" platforms is meaningful for addressing challenges posed by land scarcity and climate change to the supply of dietary sugar. However, upcycling CO2 into complex polyoxygenated carbohydrates involves several major challenges, including achieving enantioselective and thermodynamically driven transformation and expanding product repertoires while reducing energy consumption. We present a versatile chemoenzymatic roadmap based on aldol condensation, iso/epimerization, and dephosphorylation reactions for asymmetric CO2 and H2 assembly into sugars with perfect stereocontrol. In particular, we developed a minimum ATP consumption and the shortest pathway for bottom-up biosynthesis of the fundamental precursor, fructose-6-phosphate, which is valuable for synthesizing structure-diverse sugars and derivatives. Engineering bottleneck-associated enzyme catalysts aided in the thermodynamically driven synthesis of several energy-dense and functional hexoses, such as glucose and D-allulose, featuring higher titer (63 mmol L-1) and CO2-product conversion rates (25 mmol C L-1 h-1) compared to established in vitro CO2-fixing pathways. This chemical-biological platform demonstrated greater carbon conversion yield than the conventional "CO2-bioresource-sugar" process and could be easily extended to precisely synthesize other high-order sugars from CO2.

Preoperative CT-guided localization of pulmonary nodules with low-dose radiation.

Background: Video-assisted thoracoscopic surgery (VATS) has been widely accepted for the treatment of pulmonary nodules. Prior to VATS, pulmonary nodules can be labeled by computed tomography (CT)-guided hook wire localization, but multiple scans are required, which increases the total radiation dose. We aimed to assess the effectiveness and risks of using low-dose radiation CT to locate lung nodules prior to VATS. Methods: This study included 158 patients who underwent VATS resection after CT-guided hook wire localization. Based on the CT tube voltage, patients were split into two groups: the low-voltage group (Group A) received 80 kV, while the high-voltage group (Group B) received 120 kV. The two groups' image quality, radiation exposure, localization success and complication rates were compared. The frequencies of intraoperative complications and the types of lung nodules were also compared between the groups. Results: Successful nodule mapping was obtained in 158 patients. There was no significant difference in age, sex ratio or BMI between the two groups. Subjective imaging quality in both groups met the requirements for location (≥2 points). The signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) in Group A were lower than those in Group B (P<0.05). Furthermore, the dose length product (DLP) and effective dose (ED) in Group A were lower than those in Group B (P<0.05). Conclusions: Low-dose radiation CT-guided localization is safe and feasible for identifying uncertain pulmonary nodules before VATS, enabling a significant radiation dose reduction while maintaining mapping accuracy and not increasing complication risk.

Functional Analysis of Novel alkB Genes Encoding Long-Chain n-Alkane Hydroxylases in Rhodococcus sp. Strain CH91.

Rhodococcus sp. strain CH91 is capable of utilizing long-chain n-alkanes as the sole carbon source. Two new genes (alkB1 and alkB2) encoding AlkB-type alkane hydroxylase were predicted by its whole-genome sequence analysis. The purpose of this study was to elucidate the functional role of alkB1 and alkB2 genes in the n-alkane degradation of strain CH91. RT-qPCR analyses revealed that the two genes were induced by n-alkanes ranging from C16 to C36 and the expression of the alkB2 gene was up-regulated much higher than that of alkB1. The knockout of the alkB1 or alkB2 gene in strain CH91 resulted in the obvious reduction of growth and degradation rates on C16-C36 n-alkanes and the alkB2 knockout mutant exhibited lower growth and degradation rate than the alkB1 knockout mutant. When gene alkB1 or alkB2 was heterologously expressed in Pseudomonas fluorescens KOB2Δ1, the two genes could restore its alkane degradation activity. These results demonstrated that both alkB1 and alkB2 genes were responsible for C16-C36 n-alkanes' degradation of strain CH91, and alkB2 plays a more important role than alkB1. The functional characteristics of the two alkB genes in the degradation of a broad range of n-alkanes make them potential gene candidates for engineering the bacteria used for bioremediation of petroleum hydrocarbon contaminations.

The differences between broad bean koji fermented in laboratory and factory conditions by an efficient Aspergillus oryzae.

Broad bean paste-meju was fermented by a mixture of broad bean koji and saline; koji fermentation is an essential process for the production of broad bean paste-meju. Aspergillus oryzae was the most widely used in sauce fermentation. The purpose of this study was to research the factory adaptability of the highly efficient A. oryzae PNM003 and further evaluate the effect of fermentation conditions and fermentation strains on koji. A. oryzae PNM003 was compared with the widely used strain HN 3.042 not only in the laboratory but also in factory conditions (large scale). Results showed that the koji made with the same starter in the factory had a greater amount of fungi than that in the laboratory. Bacteria and yeast levels in HN_L koji were higher than in PN_L koji. As for fungi constitution, almost only Aspergillus survived in the end through the microorganism self-purification process during koji fermentation. As for the bacterial constitution, koji was grouped by fermentation conditions instead of fermentation starter. PN koji had higher protease activity and a higher content of total acids, amino acid nitrogen, amino acids, and organic acids in the laboratory conditions. Nevertheless, in factory conditions, PN koji and HN koji had similar indexes. As for volatile flavor compounds, koji made with the two starters in the same condition was grouped together. As for the same starter, there were more flavor compounds metabolized in the factory condition than in the laboratory condition, especially esters and alcohols. The results showed PN was a highly efficient strain to ferment koji, but the advantages were expressed more remarkably in laboratory conditions. In brief, the fermented condition had a greater influence than the fermentation starter for broad bean koji.

A comparison of computed tomography imaging with histopathology in the sensitivity and correlation of evaluating coronary arterial calcification.

Background: The sensitivity and correlation of coronary computed tomography angiography (CTA) as compared with histopathology are unknown in evaluating coronary arterial calcification. In this study, we retrospectively evaluated qualitatively and quantitatively the sensitivity and correlation of coronary CTA compared with histopathology in assessing coronary arterial calcification. Methods: This study was conducted on 12 randomly selected cadavers aged over 40 years at the time of death, and 53 segments of coronary arteries from these 12 cadavers were obtained from the Human Anatomy Laboratory of Tianjin Medical University. The artery segments were scanned using contrasted-enhanced dual-source computed tomography (DSCT) with an axial slice thickness of 0.6 mm. Coronary artery calcification in a coronary segment was defined as the presence of 1 or more voxels with a CT density >130 Hounsfield units. According to the arc of calcification in the cross section of the coronary artery wall, calcified plaques were divided into three categories: mild, moderate, and severe calcification. The coronary artery stenosis caused by calcified plaque was observed and calculated with multiplanar reconstruction (MPR), maximum density projection, volume rendering (VR), and cross-sectional reconstruction. After CT enhancement scanning, the coronary artery specimens were cut into 4-mm long segments and embedded in paraffin for pathological staining. Pathological classification and coronary artery stenosis measured with pathological analysis were used as comparison criteria. Results: Histopathology detected 69 Vb-type plaques, while DSCT detected 57 calcified plaques. The sensitivity of CT for detecting mild, moderate, and severe calcified plaques were 88.3% [95% confidence interval (CI): 74.1-95.6%], 100% (95% CI: 69.8-100%), and 100% (95% CI: 73.2-100%), respectively. DSCT had a significant (P<0.001) correlation with histopathology in quantifying coronary artery stenosis caused by mild, moderate, and severe calcified plaques (R2=0.9278, R2=0.9158, R2=0.7923, respectively). Compared with histopathology, DSCT overestimated coronary artery stenosis caused by mild, moderate, and severe calcified plaques (3.2%±2.0%, 4.9%±4.7%, and 14.7%±8.2%, respectively; P<0.05). Conclusions: DSCT contrast enhancement scanning can detect and characterize coronary artery calcification with a good correlation with histopathologic quantification of coronary artery stenosis caused by different types of calcified plaques, even though coronary CTA may overestimate the stenosis.

A bioflocculant from Corynebacterium glutamicum and its application in acid mine wastewater treatment.

Although many microorganisms have been found to produce bioflocculants, and bioflocculants have been considered as attractive alternatives to chemical flocculants in wastewater treatment, there are few reports on bioflocculants from the safe strain C. glutamicum, and the application of bioflocculants in acid wastewater treatment is also rare attributed to the high content of metal ions and high acidity of the water. In this study, a novel bioflocculant produced by Corynebacterium glutamicum Cg1-P30 was investigated. An optimal production of this bioflocculant with a yield of 0.52 g/L was achieved by Box-Behnken design, using 12.20 g/L glucose, 4.00 g/L corn steep liquor and 3.60 g/L urea as carbon and nitrogen source. The structural characterization revealed that the bioflocculant was mainly composed of 37.50% neutral sugar, 10.03% uronic acid, 6.32% aminosugar and 16.51% protein. Carboxyl, amine and hydroxyl groups were the functional groups in flocculation. The biofocculant was thermally stable and dependent on metal ions and acidic pH, showing a good flocculating activity of 91.92% at the dosage of 25 mg/L by aid of 1.0 mM Fe3+ at pH 2.0. Due to these unique properties, the bioflocculant could efficiently remove metal ions such as Fe, Al, Zn, and Pb from the real acid mine wastewater sample without pH adjustment, and meanwhile made the acid mine wastewater solution become clear with an increased neutral pH. These findings suggested the great potential application of the non-toxic bioflocculant from C. glutamicum Cg1-P30 in acid mine wastewater treatment.

Biosynthesis of artificial starch and microbial protein from agricultural residue.

Growing populations and climate change pose great challenges to food security. Humankind is confronting a serious question: how will we feed the world in the near future? This study presents an out-of-the-box solution involving the highly efficient biosynthesis of artificial starch and microbial proteins from available and abundant agricultural residue as new feed and food sources. A one-pot biotransformation using an in vitro coenzyme-free synthetic enzymatic pathway and baker's yeast can simultaneously convert dilute sulfuric acid-pretreated corn stover to artificial starch and microbial protein under aerobic conditions. The ß-glucosidase-free commercial cellulase mixture plus an ex vivo two-enzyme complex containing cellobiose phosphorylase and potato α-glucan phosphorylase displayed on the surface of Saccharomyces cerevisiae, showed better cellulose hydrolysis rates than a commercial ß-glucosidase-rich cellulase mixture. This is because the channeling of the hydrolytic product from the solid cellulosic feedstock to the yeast mitigated the inhibition of the cellulase cocktail. Animal tests have shown that the digestion of artificial amylose results in slow and relatively small changes in blood sugar levels, suggesting that it could be a new health food component that prevents obesity and diabetes. A combination of the utilization of available agricultural residue and the biosynthesis of starch and microbial protein from non-food biomass could address the looming food crisis in the food-energy-water nexus.

Feasibility study of using low-kilovoltage, prospective gating, high-pitch, dual-source computed tomography prior to transcatheter aortic valve replacement: analysis of image quality and radiation dose.

Background: Computed tomography (CT) scans before transcatheter aortic valve replacement (TAVR) are used to evaluate the aortic valve and guide the selection of appropriate valve stents. Accurate imaging evaluation can ensure the success rate of surgery while reducing the incidence of complications. Multiple studies have adopted a protocol of coronary artery, aortic valve, and total aortic scan, with the patients receiving higher radiation doses. The aim of this study was to evaluate the image quality, radiation dose, and diagnostic performance of dual-source computed tomography (DSCT) with high-pitch spiral scanning for TAVR. Methods: A total of 240 patients being evaluated for TAVR were continuously enrolled. Based on the differences in electrocardiography (ECG) gating and tube voltage, the patients were divided into 4 groups: group A, 70-kV prospective ECG gating, high-pitch helical; group B, 70-kV retrospective ECG gating; group C, 100-kV prospective ECG gating, high-pitch helical; and group D, 120-kV prospective ECG gating, high-pitch helical. Image quality was evaluated on a 4-point scale. The image signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) were calculated for objective evaluation. The radiation doses of all patients were recorded. The image quality and radiation dose of each group were compared. Results: There were no differences in age, body mass index (BMI), subjective image quality scores, CT values between the aorta and the coronary artery, or image CNR between the 4 groups. The mean radiation doses of groups A-D were 4.13±0.69, 4.79±0.58, 12.00±1.62, and 15.01±1.90 mSv, respectively. The mean radiation dose in group A (70-kV prospective ECG gating) decreased significantly (P<0.05). Conclusions: Using low-kilovoltage, high-pitch DSCT can provide comparable image quality for TAVR evaluation and significantly reduce the radiation dose.

Microbial (E)-4-hydroxy-3-methylbut-2-enyl pyrophosphate reductase (IspH) and its biotechnological potential: A mini review.

(E)-4-hydroxy-3-methylbut-2-enyl pyrophosphate (HMBPP) reductase (IspH) is a [4Fe-4S] cluster-containing enzyme, involved in isoprenoid biosynthesis as the final enzyme of the methylerythritol phosphate (MEP) pathway found in many bacteria and malaria parasites. In recent years, many studies have revealed that isoprenoid compounds are an alternative to petroleum-derived fuels. Thus, ecofriendly methods harnessing the methylerythritol phosphate pathway in microbes to synthesize isoprenoid compounds and IspH itself have received notable attention from researchers. In addition to its applications in the field of biosynthesis, IspH is considered to be an attractive drug target for infectious diseases such as malaria and tuberculosis due to its survivability in most pathogenic bacterium and its absence in humans. In this mini-review, we summarize previous reports that have systematically illuminated the fundamental and structural properties, substrate binding and catalysis, proposed catalytic mechanism, and novel catalytic activities of IspH. Potential bioengineering and biotechnological applications of IspH are also discussed.

An efficient CRISPR/Cas9-based genome editing system for alkaliphilic Bacillus sp. N16-5 and application in engineering xylose utilization for D-lactic acid production.

Alkaliphiles are considered more suitable chassis than traditional neutrophiles due to their excellent resistance to microbial contamination. Alkaliphilic Bacillus sp. N16-5, an industrially interesting strain with great potential for the production of lactic acid and alkaline polysaccharide hydrolases, can only be engineered genetically by the laborious and time-consuming homologous recombination. In this study, we reported the successful development of a CRISPR/Cas9-based genome editing system with high efficiency for single-gene deletion, large gene fragment deletion and exogenous DNA chromosomal insertion. Moreover, based on a catalytically dead variant of Cas9 (dCas9), we also developed a CRISPRi system to efficiently regulate gene expression. Finally, this efficient genome editing system was successfully applied to engineer the xylose metabolic pathway for the efficient bioproduction of D-lactic acid. Compared with the wild-type Bacillus sp. N16-5, the final engineered strain with XylR deletion and AraE overexpression achieved 34.3% and 27.7% increases in xylose consumption and D-lactic acid production respectively. To our knowledge, this is the first report on the development and application of CRISPR/Cas9-based genome editing system in alkaliphilic Bacillus, and this study will significantly facilitate functional genomic studies and genome manipulation in alkaliphilic Bacillus, laying a foundation for the development of more robust microbial chassis.

Asymmetric Synthesis of Fused-Ring Tetrahydroisoquinolines and Tetrahydro-ß-carbolines from 2-Arylethylamines via a Chemoenzymatic Approach.

While chiral fused-ring tetrahydroisoquinoline (THIQ) and tetrahydro-ß-carboline (THßC) scaffolds have attracted considerable interest due to their wide spectrum of biological activities, the synthesis of optically pure chiral fused-ring THIQs and THßCs remains a challenging task. Herein, a group of active imine reductases were identified to convert the imine precursors into the corresponding enantiocomplementary fused-ring THIQs and THßCs with high enantioselectivity and conversion, establishing an efficient and green chemoenzymatic approach to fused-ring alkaloids from 2-arylethylamines.

Engineering substrate specificity of HAD phosphatases and multienzyme systems development for the thermodynamic-driven manufacturing sugars.

Naturally, haloacid dehalogenase superfamily phosphatases have been evolved with broad substrate promiscuity; however, strong specificity to a particular substrate is required for developing thermodynamically driven routes for manufacturing sugars. How to alter the intrinsic substrate promiscuity of phosphatases and fit the "one enzyme-one substrate" model remains a challenge. Herein, we report the structure-guided engineering of a phosphatase, and successfully provide variants with tailor-made preference for three widespread phosphorylated sugars, namely, glucose 6-phosphate, fructose 6-phosphate, and mannose 6-phosphate, while simultaneously enhancement in catalytic efficiency. A 12000-fold switch from unfavorite substrate to dedicated one is generated. Molecular dynamics simulations reveal the origin of improved activity and substrate specificity. Furthermore, we develop four coordinated multienzyme systems and accomplish the conversion of inexpensive sucrose and starch to fructose and mannose in excellent yield of 94-96%. This innovative sugar-biosynthesis strategy overcomes the reaction equilibrium of isomerization and provides the promise of high-yield manufacturing of other monosaccharides and polyols.

Degradation of long-chain n-alkanes by a novel thermal-tolerant Rhodococcus strain.

A novel bacterial strain, CH91, was isolated from a high-temperature oil reservoir. Morphological characterization, phylogenetic analyses of 16S rRNA gene sequence and genome relatedness indicated that the strain is a potential new species in the genus Rhodococcus. Strain CH91 could grow in the temperature range of 25-50 °C (optimally at 37 °C) and utilize a broad range of long-chain n-alkanes from hexadecane to hexatriacontane. The utilization of the n-alkanes mixture of strain CH91 revealed that the degradation rate was correlated to the length of the carbon chain. Two novel alkB genes encoding alkane 1-monooxygenase were found in the genome of this strain. The protein sequences of both alkane 1-monooxygenases showed a remarkable phylogenetic distance to other reported AlkB protein sequences. These results would help broaden our knowledge about alkane degradation by Rhodocuccus and its potential ecological role. The ability of the strain in the long-chain alkane degradation and thermal tolerance could also be further exploited for bioremediation of oil contaminations and microbial enhanced oil recovery.

Reproducibility of radiomic features of pulmonary nodules between low-dose CT and conventional-dose CT.

Background: The reproducibility of radiomic features is essential to lung cancer detection. This study aimed to investigate the reproducibility of radiomic features of pulmonary nodules between low-dose computed tomography (LDCT) and conventional-dose computed tomography (CDCT). Methods: A total of 105 patients with 119 pulmonary nodules [39 ground-glass nodules (GGNs) and 80 solid nodules] who underwent LDCT and CDCT were retrospectively studied between September 2019 and November 2020. Pulmonary nodules were manually segmented and 1,125 radiomic features (shape, first-order intensity, texture, wavelet, and Laplacian of the Gaussian features) were extracted from both LDCT and CDCT images. The concordance correlation coefficient (CCC) was used to evaluate the reproducibility of these radiomic features. Results: Of the 1,125 radiomic features considered, 35.5% (399 of 1,125) and 41.5% (467 of 1,125) were reproducible (CCC ≥0.85) for GGNs and solid nodules, respectively. The intensity, texture, and wavelet features of solid nodules were more reproducible than those of GGNs. The mean CCC values for intensity and texture features of solid nodules were of 0.85 and above, whereas the mean values for those of GGNs were of less than 0.85. After Gaussian kernel (σ =2) preprocessing, the CCC of intensity and texture features of GGNs improved from 0.77 to 0.90, and 84.9% (79 of 93) of the radiomic features were reproducible (mean CCC increase from 0.84±0.13 to 0.92±0.08 for intensity features, and from 0.75±0.15 to 0.89±0.11 for texture features). Wavelet features had the lowest CCCs for both GGNs and solid nodules. Conclusions: The majority of the radiomic feature classes of solid pulmonary nodules have a high level of reproducibility between LDCT and CDCT. However, LDCT should not be used as an alternative to CDCT in the radiomic study of GGNs.

Imperfect guide-RNA (igRNA) enables CRISPR single-base editing with ABE and CBE.

CRISPR base editing techniques tend to edit multiple bases in the targeted region, which is a limitation for precisely reverting disease-associated single-nucleotide polymorphisms (SNPs). We designed an imperfect gRNA (igRNA) editing methodology, which utilized a gRNA with one or more bases that were not complementary to the target locus to direct base editing toward the generation of a single-base edited product. Base editing experiments illustrated that igRNA editing with CBEs greatly increased the single-base editing fraction relative to normal gRNA editing with increased editing efficiencies. Similar results were obtained with an adenine base editor (ABE). At loci such as DNMT3B, NSD1, PSMB2, VIATA hs267 and ANO5, near-perfect single-base editing was achieved. Normally an igRNA with good single-base editing efficiency could be selected from a set of a few igRNAs, with a simple protocol. As a proof-of-concept, igRNAs were used in the research to construct cell lines of disease-associated SNP causing primary hyperoxaluria construction research. This work provides a simple strategy to achieve single-base base editing with both ABEs and CBEs and overcomes a key obstacle that limits the use of base editors in treating SNP-associated diseases or creating disease-associated SNP-harboring cell lines and animal models.

A New 3-Ketosteroid-Δ1-Dehydrogenase with High Activity and Broad Substrate Scope for Efficient Transformation of Hydrocortisone at High Substrate Concentration.

3-Ketosteroid-Δ1-dehydrogenases (KstDs [EC 1.3.99.4]) catalyze the Δ1-dehydrogenation of steroids and are a class of important enzymes for steroid biotransformations. In this study, nine putative kstD genes from different origins were selected and overexpressed in Escherichia coli BL21(DE3). These recombinant enzymes catalyzed the Δ1-desaturation of a variety of steroidal compounds. Among them, the KstD from Propionibacterium sp. (PrKstD) displayed the highest specific activity and broad substrate spectrum. The detailed catalytic characterization of PrKstD showed that it can convert a wide range of 3-ketosteroid compounds with diverse substituents, ranging from substituents at the C9, C10, C11 and C17 position through substrates without C4-C5 double bond, to previously inactive C6-substituted ones such as 11ß,17-dihydroxy-6α-methyl-pregn-4-ene-3,20-dione. Reaction conditions were optimized for the biotransformation of hydrocortisone in terms of pH, temperature, co-solvent and electron acceptor. By using 50 g/L wet resting E. coli cells harboring PrKstD enzyme, the conversion of hydrocortisone was about 92.5% within 6 h at the substrate concentration of 80 g/L, much higher than the previously reported results, demonstrating the application potential of this new KstD.