Evaluating the potential of different bioaugmented strains to enhance methane production during thermophilic anaerobic digestion of food waste.

Bioaugmentation technology for improving the performance of thermophilic anaerobic digestion (TAD) of food waste (FW) treatment is gaining more attention. In this study, four thermophilic strains (Ureibacillus suwonensis E11, Clostridium thermopalmarium HK1, Bacillus thermoamylovorans Y25 and Caldibacillus thermoamylovorans QK5) were inoculated in the TAD of FW system, and the biochemical methane potential (BMP) batch study was conducted to assess the potential of different bioaugmented strains to enhance methane production. The results showed that the cumulative methane production in groups inoculated with E11, HK1, Y25 and QK5 improved by 2.05%, 14.54%, 19.79% and 9.17%, respectively, compared with the control group with no inoculation. Moreover, microbial community composition analysis indicated that the relative abundance of the main hydrolytic bacteria and/or methanogenic archaea was increased after bioaugmentation, and the four strains successfully became representative bacterial biomarkers in each group. The four strains enhanced methane production by strengthening starch, sucrose, galactose, pyruvate and methane metabolism functions. Further, the correlation networks demonstrated that the representative bacterial genera had positive correlations with the differential metabolic functions in each bioaugmentation group. This study provides new insights into the TAD of FW with bioaugmented strains.

Metagenome reveals the possible mechanism that microbial strains promote methanogenesis during anaerobic digestion of food waste.

For better understanding the mechanism of microbial strains promoting methane production, four strains Hungatella xylanolytica A5, Bacillus licheniformis B1, Paraclostridium benzoelyticum C2 and Advenella faeciporci E1 were inoculated into anaerobic digestion systems. After bioaugmentation, the cumulative methane production of A5, B1, C2 and E1 groups elevated by 11.68%, 8.20%, 18.21% and 15.67% compared to CK group, respectively. The metagenomic analysis revealed that the species diversity and uniformity of the experimental groups was improved, and hydrolytic acidifying bacteria, represented by Clostridiaceae, Anaerolineaceae and Oscillospiraceae, and methanogens, such as Methanotrichaceae and Methanobacteriaceae, were enriched. Meanwhile, the abundance of key genes in carbohydrate, pyruvate and methane metabolism was increased in the inoculated groups, providing reasonable reasons for more methane production. The strengthening mechanism of microbial strains in this study offered a theoretical foundation for selecting a suitable bioaugmentation strategy to solve the problems of slow start-up and low methane production in anaerobic digestion.

Improving the production of 9α-hydroxy-4-androstene-3,17-dione from phytosterols by 3-ketosteroid-Δ1-dehydrogenase deletions and multiple genetic modifications in Mycobacterium fortuitum.

BACKGROUND: 9α-hydroxyandrost-4-ene-3,17-dione (9-OHAD) is a significant intermediate for the synthesis of glucocorticoid drugs. However, in the process of phytosterol biotransformation to manufacture 9-OHAD, product degradation, and by-products restrict 9-OHAD output. In this study, to construct a stable and high-yield 9-OHAD producer, we investigated a combined strategy of blocking Δ1­dehydrogenation and regulating metabolic flux. RESULTS: Five 3-Ketosteroid-Δ1-dehydrogenases (KstD) were identified in Mycobacterium fortuitum ATCC 35855. KstD2 showed the highest catalytic activity on 3-ketosteroids, followed by KstD3, KstD1, KstD4, and KstD5, respectively. In particular, KstD2 had a much higher catalytic activity for C9 hydroxylated steroids than for C9 non-hydroxylated steroids, whereas KstD3 showed the opposite characteristics. The deletion of kstDs indicated that KstD2 and KstD3 were the main causes of 9-OHAD degradation. Compared with the wild type M. fortuitum ATCC 35855, MFΔkstD, the five kstDs deficient strain, realized stable accumulation of 9-OHAD, and its yield increased by 42.57%. The knockout of opccr or the overexpression of hsd4A alone could not reduce the metabolic flux of the C22 pathway, while the overexpression of hsd4A based on the knockout of opccr in MFΔkstD could remarkably reduce the contents of 9,21 ­dihydroxy­20­methyl­pregna­4­en­3­one (9-OHHP) by-products. The inactivation of FadE28-29 leads to a large accumulation of incomplete side-chain degradation products. Therefore, hsd4A and fadE28-29 were co-expressed in MFΔkstDΔopccr successfully eliminating the two by-products. Compared with MFΔkstD, the purity of 9-OHAD improved from 80.24 to 90.14%. Ultimately, 9­OHAD production reached 12.21 g/L (83.74% molar yield) and the productivity of 9-OHAD was 0.0927 g/L/h from 20 g/L phytosterol. CONCLUSIONS: KstD2 and KstD3 are the main dehydrogenases that lead to 9-OHAD degradation. Hsd4A and Opccr are key enzymes regulating the metabolic flux of the C19- and C22-pathways. Overexpression of fadE28-29 can reduce the accumulation of incomplete degradation products of the side chains. According to the above findings, the MF-FA5020 transformant was successfully constructed to rapidly and stably accumulate 9-OHAD from phytosterols. These results contribute to the understanding of the diversity and complexity of steroid catabolism regulation in actinobacteria and provide a theoretical basis for further optimizing industrial microbial catalysts.

Rational design of GDP­D­mannose mannosyl hydrolase for microbial L­fucose production.

BACKGROUND: L­Fucose is a rare sugar that has beneficial biological activities, and its industrial production is mainly achieved with brown algae through acidic/enzymatic fucoidan hydrolysis and a cumbersome purification process. Fucoidan is synthesized through the condensation of a key substance, guanosine 5'­diphosphate (GDP)­L­fucose. Therefore, a more direct approach for biomanufacturing L­fucose could be the enzymatic degradation of GDP­L­fucose. However, no native enzyme is known to efficiently catalyze this reaction. Therefore, it would be a feasible solution to engineering an enzyme with similar function to hydrolyze GDP­L­fucose. RESULTS: Herein, we constructed a de novo L­fucose synthetic route in Bacillus subtilis by introducing heterologous GDP­L­fucose synthesis pathway and engineering GDP­mannose mannosyl hydrolase (WcaH). WcaH displays a high binding affinity but low catalytic activity for GDP­L­fucose, therefore, a substrate simulation­based structural analysis of the catalytic center was employed for the rational design and mutagenesis of selected positions on WcaH to enhance its GDP­L­fucose­splitting efficiency. Enzyme mutants were evaluated in vivo by inserting them into an artificial metabolic pathway that enabled B. subtilis to yield L­fucose. WcaHR36Y/N38R was found to produce 1.6 g/L L­fucose during shake­flask growth, which was 67.3% higher than that achieved by wild­type WcaH. The accumulated L­fucose concentration in a 5 L bioreactor reached 6.4 g/L. CONCLUSIONS: In this study, we established a novel microbial engineering platform for the fermentation production of L­fucose. Additionally, we found an efficient GDP­mannose mannosyl hydrolase mutant for L­fucose biosynthesis that directly hydrolyzes GDP­L­fucose. The engineered strain system established in this study is expected to provide new solutions for L­fucose or its high value­added derivatives production.

Phytosterol conversion into C9 non-hydroxylated derivatives through gene regulation in Mycobacterium fortuitum.

Androst-4-ene-3,17-dione (AD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) are important drug intermediates that can be biosynthesized from phytosterols. However, the C9 hydroxylation of steroids via 3-ketosteroid 9α-hydroxylase (KSH) limits AD and 4-HBC accumulation. Five active KshAs, the oxidation component of KSH, were identified in Mycobacterium fortuitum ATCC 35855 for the first time. The deletion of kshAs indicated that the five KshA genes were jointly responsible for C9 hydroxylation during phytosterol biotransformation. MFKDΔkshA, the five KshAs deficient strain, blocked C9 hydroxylation and produced 5.37 g/L AD and 0.55 g/L 4-HBC. The dual function reductase Opccr knockout and 17ß-hydroxysteroid dehydrogenase Hsd4A enhancement reduced 4-HBC content from 8.75 to 1.72% and increased AD content from 84.13 to 91.34%, with 8.24 g/L AD being accumulated from 15 g/L phytosterol. In contrast, hsd4A and thioesterase fadA5 knockout resulted in the accumulation of 5.36 g/L 4-HBC from 10 g/L phytosterol. We constructed efficient AD (MFKDΔkshAΔopccr_hsd4A) and 4-HBC (MFKDΔkshAΔhsd4AΔfadA5) producers and provided insights for further metabolic engineering of the M. fortuitum ATCC 35855 strain for steroid productions. KEY POINTS: • Five active KshAs were first identified in M. fortuitum ATCC 35855. • Deactivation of all five KshAs blocks the steroid C9 hydroxylation reaction. • AD or 4-HBC production was improved by Hsd4A, FadA5, and Opccr modification.

Production of 21-hydroxy-20-methyl-pregna-1,4-dien-3-one by modifying multiple genes in Mycolicibacterium.

C22 steroid drug intermediates are suitable for corticosteroids synthesis, and the production of C22 steroids is unsatisfactory due to the intricate steroid metabolism. Among the C22 steroids, 21-hydroxy-20-methyl-pregna-1,4-dien-3-one (1,4-HP) could be used for Δ1-steroid drug synthesis, such as prednisolone. Nevertheless, the production of 1,4-HP remains unsatisfactory. In this study, an ideal 1,4-HP producing strain was constructed. By the knockout of 3-ketosteroid-9-hydroxylase (KshA) genes and 17ß-hydroxysteroid dehydrogenase (Hsd4A) gene, the steroid nucleus degradation and the accumulation of C19 steroids in Mycolicibacterium neoaurum were blocked. The mutant strain could transform phytosterols into 1,4-HP as the main product and 21-hydroxy-20-methyl-pregna-4-ene-3-one as a by-product. Subsequently, the purity of 1,4-HP improved to 95.2% by the enhancement of 3-ketosteroid-Δ1-dehydrogenase (KSTD) activity, and the production of 1,4-HP was improved by overexpressing NADH oxidase (NOX) and catalase (KATE) genes. Consequently, the yield of 1,4-HP achieved 10.5 g/L. The molar yield and the purity of 1,4-HP were optimal so far, and the production of 1,4-HP provides a new intermediate for the pharmaceutical steroid industry. KEY POINTS: • A third 3-ketosteroid-9-hydroxylase was identified in Mycolicibacterium neoaurum. • An 1,4-HP producer was constructed by KshA and Hsd4A deficiency. • The production of 1,4-HP was improved by KSTD, NOX, and KATE overexpression.

Functional analysis of acyl-CoA dehydrogenases and their application to C22 steroid production.

Acyl-CoA dehydrogenase (ChsE) is involved in the steroid side-chain degradation process. However, their function in vivo remains unclear. In this study, three ChsE, ChsE1-ChsE2, ChsE3, and ChsE4-ChsE5, were identified in Mycolicibacterium neoaurum, and their functions in vivo are studied and compared with those from Mycobacterium tuberculosis in vitro. By gene knockout, complementation, and the bioconversion of phytosterols, the function of ChsE was elucidated that ChsE4-ChsE5 could utilize C27, C24, and C22 steroids in vivo. ChsE3 could utilize C27 and C24 steroids in vivo. ChsE1-ChsE2 could utilize C27, C24, and C22 steroids in vivo. What is more, the production strain of a C22 steroid, 3-oxo-4,17-pregadiene-20-carboxylic acid methyl ester (PDCE), is constructed with ChsE overexpression. This study improved the understanding of the steroid bioconversion pathway and proposed a method of the production of a new C22 steroid. KEY POINTS: • Three ChsE paralogs from M. neoaurum are identified and studied. • The function of ChsE is overlapped in vivo. • A C22 steroid (PDCE) producer was constructed with ChsE overexpression.

Whole-Genome Analysis of Mycobacterium neoaurum DSM 1381 and the Validation of Two Key Enzymes Affecting C22 Steroid Intermediates in Sterol Metabolism.

Mycobacterium neoaurum DSM 1381 originated from Mycobacterium neoaurum ATCC 25790 by mutagenesis screening is a strain of degrading phytosterols and accumulating important C22 steroid intermediates, including 22-hydroxy-23, 24-bisnorchola-4-en-3-one (4-HP) and 22-hydroxy-23, 24-bisnorchola-1,4-dien-3-one (HPD). However, the metabolic mechanism of these C22 products in M. neoaurum DSM 1381 remains unknown. Therefore, the whole-genome sequencing and comparative genomics analysis of M. neoaurum DSM 1381 and its parent strain M. neoaurum ATCC 25790 were performed to figure out the mechanism. As a result, 28 nonsynonymous single nucleotide variants (SNVs), 17 coding region Indels, and eight non-coding region Indels were found between the genomes of the two strains. When the wild-type 3-ketosteroid-9α-hydroxylase subunit A1 (KshA1) and ß-hydroxyacyl-CoA dehydrogenase (Hsd4A) were overexpressed in M. neoaurum DSM 1381, the steroids were transformed into the 4-androstene-3, 17- dione (AD) and 1,4-androstadiene-3,17-dione (ADD) instead of C22 intermediates. This result indicated that 173N of KshA1 and 171K of Hsd4A are indispensable to maintaining their activity, respectively. Amino acid sequence alignment analysis show that both N173D in KshA1 and K171E in Hsd4A are conservative sites. The 3D models of these two enzymes were predicted by SWISS-MODEL and AlphaFold2 to understand the inactivation of the two key enzymes. These results indicate that K171E in Hsd4A may destroy the inaction between the NAD+ with the NH3+ and N173D in KshA1 and may disrupt the binding of the catalytic domain to the substrate. A C22 steroid intermediates-accumulating mechanism in M. neoaurum DSM 1381 is proposed, in which the K171E in Hsd4A leads to the enzyme's inactivation, which intercepts the C19 sub-pathways and accelerates the C22 sub-pathways, and the N173D in KshA1 leads to the enzyme's inactivation, which blocks the degradation of C22 intermediates. In conclusion, this study explained the reasons for the accumulation of C22 intermediates in M. neoaurum DSM 1381 by exploring the inactivation mechanism of the two key enzymes.

Bioconversion of Phytosterols to 9-Hydroxy-3-Oxo-4,17-Pregadiene-20-Carboxylic Acid Methyl Ester by Enoyl-CoA Deficiency and Modifying Multiple Genes in Mycolicibacterium neoaurum.

Steroid drug precursors, including C19 and C22 steroids, are crucial to steroid drug synthesis and development. However, C22 steroids are less developed due to the intricacy of the steroid metabolic pathway. In this study, a C22 steroid drug precursor, 9-hydroxy-3-oxo-4,17-pregadiene-20-carboxylic acid methyl ester (9-OH-PDCE), was successfully obtained from Mycolicibacterium neoaurum by 3-ketosteroid-Δ1-dehydrogenase and enoyl-CoA hydratase ChsH deficiency. The production of 9-OH-PDCE was improved by the overexpression of 17ß-hydroxysteroid dehydrogenase Hsd4A and acyl-CoA dehydrogenase ChsE1-ChsE2 to reduce the accumulation of by-products. The purity of 9-OH-PDCE in fermentation broth was improved from 71.7% to 89.7%. Hence, the molar yield of 9-OH-PDCE was improved from 66.7% to 86.7%, with a yield of 0.78 g/L. Furthermore, enoyl-CoA hydratase ChsH1-ChsH2 was identified to form an indispensable complex in Mycolicibacterium neoaurum DSM 44704. IMPORTANCE C22 steroids are valuable precursors for steroid drug synthesis, but the development of C22 steroids remains unsatisfactory. This study presented a strategy for the one-step bioconversion of phytosterols to a C22 steroid drug precursor, 9-hydroxy-3-oxo-4,17-pregadiene-20-carboxylic acid methyl ester (9-OH-PDCE), by 3-ketosteroid-Δ1-dehydrogenase and enoyl-CoA hydratase deficiency with overexpression of 17ß-hydroxysteroid dehydrogenase acyl-CoA dehydrogenase in Mycolicibacterium. The function of the enoyl-CoA hydratase ChsH in vivo was revealed. Construction of the novel C22 steroid drug precursor producer provided more potential for steroid drug synthesis, and the characterization of the function of ChsH and the transformation of steroids further revealed the steroid metabolic pathway.

A Study of Type II ɛ-PL Degrading Enzyme (pldII) in Streptomyces albulus through the CRISPRi System.

ε-Poly-L-lysine (ε-PL) is a widely used antibacterial peptide polymerized of 25-35 L-lysine residues. The antibacterial effect of ε-PL is closely related to the polymerization degree. However, the mechanism of ε-PL degradation in S. albulus remains unclear. This study utilized the integrative plasmid pSET152-based CRISPRi system to transcriptionally repress the ε-PL degrading enzyme (pldII). The expression of pldII is regulated by changing the recognition site of dCas9. Through the ε-PL bacteriostatic experiments of repression strains, it was found that the repression of pldII improves the antibacterial effect of the ε-PL product. The consecutive MALDI-TOF-MS results confirmed that the molecular weight distribution of the ε-PL was changed after repression. The repression strain S1 showed a particular peak with a polymerization degree of 44, and other repression strains also generated ε-PL with a polymerization degree of over 40. Furthermore, the homology modeling and substrate docking of pldII, a typical endo-type metallopeptidase, were performed to resolve the degradation mechanism of ε-PL in S. albulus. The hydrolysis of ε-PL within pldII, initiated from the N-terminus by two amino acid-binding residues, Thr194 and Glu281, led to varying levels of polymerization of ε-PL.

Blocking the 2,3-butanediol synthesis pathway of Klebsiella pneumoniae resulted in L-valine production.

Klebsiella pneumoniae is a 2,3-butanediol producing bacterium. Nevertheless, a design and construction of L-valine production strain was studied in this paper. The first step of 2,3-butanediol synthesis and branched-chain amino acid synthesis pathways share the same step of α-acetolactate synthesis from pyruvate. However, the two pathways are existing in parallel and do not interfere with each other in the wild-type strain. A knockout of budA blocked the 2,3-butanediol synthesis pathway and resulted in the L-valine production. The budA coded an α-acetolactate decarboxylase and catalyzed the acetoin formation from α-acetolactate. Furthermore, blocking the lactic acid synthesis by knocking out of ldhA, which is encoding a lactate dehydrogenase, improved the L-valine synthesis. 2-Ketoisovalerate is the precursor of L-valine, it is also an intermediate of the isobutanol synthesis pathway, while indole-3-pyruvate decarboxylase (ipdC) is responsible for isobutyraldehyde formation from 2-ketoisovalerate. Production of L-valine has been improved by knocking out of ipdC. On the other side, the ilvE, encoding a transaminase B, reversibly transfers one amino group from glutamate to α-ketoisovalerate. Overexpression of ilvE exhibited a distinct improvement of L-valine production. The brnQ encodes a branched-chain amino acid transporter, and L-valine production was further improved by disrupting brnQ. It is also revealed that weak acidic and aerobic conditions favor L-valine production. Based on these findings, L-valine production by metabolically engineered K. pneumonia was examined. In fed-batch fermentation, 22.4 g/L of L-valine was produced by the engineered K. pneumoniae ΔbudA-ΔldhA-ΔipdC-ΔbrnQ-ilvE after 55 h of cultivation, with a substrate conversion ratio of 0.27 mol/mol glucose.

Redirection of the central metabolism of Klebsiella pneumoniae towards dihydroxyacetone production.

BACKGROUND: Klebsiella pneumoniae is a bacterium that can be used as producer for numerous chemicals. Glycerol can be catabolised by K. pneumoniae and dihydroxyacetone is an intermediate of this catabolism pathway. Here dihydroxyacetone and glycerol were produced from glucose by this bacterium based a redirected glycerol catabolism pathway. RESULTS: tpiA, encoding triosephosphate isomerase, was knocked out to block the further catabolism of dihydroxyacetone phosphate in the glycolysis. After overexpression of a Corynebacterium glutamicum dihydroxyacetone phosphate dephosphorylase (hdpA), the engineered strain produced remarkable levels of dihydroxyacetone (7.0 g/L) and glycerol (2.5 g/L) from glucose. Further increase in product formation were obtained by knocking out gapA encoding an iosenzyme of glyceraldehyde 3-phosphate dehydrogenase. There are two dihydroxyacetone kinases in K. pneumoniae. They were both disrupted to prevent an inefficient reaction cycle between dihydroxyacetone phosphate and dihydroxyacetone, and the resulting strains had a distinct improvement in dihydroxyacetone and glycerol production. pH 6.0 and low air supplement were identified as the optimal conditions for dihydroxyacetone and glycerol production by K, pneumoniae ΔtpiA-ΔDHAK-hdpA. In fed batch fermentation 23.9 g/L of dihydroxyacetone and 10.8 g/L of glycerol were produced after 91 h of cultivation, with the total conversion ratio of 0.97 mol/mol glucose. CONCLUSIONS: This study provides a novel and highly efficient way of dihydroxyacetone and glycerol production from glucose.

Production of 9,21-dihydroxy-20-methyl-pregna-4-en-3-one from phytosterols in Mycobacterium neoaurum by modifying multiple genes and improving the intracellular environment.

BACKGROUND: Steroid drugs are essential for disease prevention and clinical treatment. However, due to intricated steroid structure, traditional chemical methods are rarely implemented into the whole synthetic process for generating steroid intermediates. Novel steroid drug precursors and their ideal bacterial strains for industrial production have yet to be developed. Among these, 9,21-dihydroxy-20-methyl-pregna-4-en-3-one (9-OH-4-HP) is a novel steroid drug precursor, suitable for the synthesis of corticosteroids. In this study, a combined strategy of blocking Δ1-dehydrogenation and the C19 pathway as well as improving the intracellular environment was investigated to construct an effective 9-OH-4-HP-producing strain. RESULTS: The Δ1-dehydrogenation-deficient strain of wild-type Mycobacterium neoaurum DSM 44074 produces 9-OH-4-HP with a molar yield of 4.8%. Hsd4A, encoding a ß-hydroxyacyl-CoA dehydrogenase, and fadA5, encoding an acyl-CoA thiolase, were separately knocked out to block the C19 pathway in the Δ1-dehydrogenation-deficient strain. The two engineered strains were able to accumulate 0.59 g L-1 and 0.47 g L-1 9-OH-4-HP from 1 g L-1 phytosterols, respectively. Furthermore, hsd4A and fadA5 were knocked out simultaneously in the Δ1-dehydrogenation-deficient strain. The 9-OH-4-HP production from the Hsd4A and FadA5 deficient strain was 11.9% higher than that of the Hsd4A deficient strain and 40.4% higher than that of the strain with FadA5 deficiency strain, respectively. The purity of 9-OH-4-HP obtained from the Hsd4A and FadA5 deficient strain has reached 94.9%. Subsequently, the catalase katE from Mycobacterium neoaurum and an NADH oxidase, nox, from Bacillus subtilis were overexpressed to improve the intracellular environment, leading to a higher 9-OH-4-HP production. Ultimately, 9-OH-4-HP production reached 3.58 g L-1 from 5 g L-1 phytosterols, and the purity of 9-OH-4-HP improved to 97%. The final 9-OH-4-HP production strain showed the best molar yield of 85.5%, compared with the previous reported strain with 30% molar yield of 9-OH-4-HP. CONCLUSION: KstD, Hsd4A, and FadA5 are key enzymes for phytosterol side-chain degradation in the C19 pathway. Double deletion of hsd4A and fadA5 contributes to the blockage of the C19 pathway. Improving the intracellular environment of Mycobacterium neoaurum during phytosterol bioconversion could accelerate the conversion process and enhance the productivity of target sterol derivatives.

1,2-Propanediol production from glycerol via an endogenous pathway of Klebsiella pneumoniae.

Klebsiella pneumoniae is an important microorganism and is used as a cell factory for many chemicals production. When glycerol was used as the carbon source, 1,3-propanediol was the main catabolite of this bacterium. K. pneumoniae ΔtpiA lost the activity of triosephosphate isomerase and prevented glycerol catabolism through the glycolysis pathway. But this strain still utilized glycerol, and 1,2-propanediol became the main catabolite. Key enzymes of 1,2-propanediol synthesis from glycerol were investigated in detail. dhaD and gldA encoded glycerol dehydrogenases were both responsible for the conversion of glycerol to dihydroxyacetone, but overexpression of the two enzymes resulted in a decrease of 1,2-propanediol production. There are two dihydroxyacetone kinases (I and II), but the dihydroxyacetone kinase I had no contribution to dihydroxyacetone phosphate formation. Dihydroxyacetone phosphate was converted to methylglyoxal, and methylglyoxal was then reduced to lactaldehyde or hydroxyacetone and further reduced to form 1,2-propanediol. Individual overexpression of mgsA, yqhD, and fucO resulted in increased production of 1,2-propanediol, but only the combined expression of mgsA and yqhD showed a positive effect on 1,2-propanediol production. The process parameters for 1,2-propanediol production by Kp ΔtpiA-mgsA-yqhD were optimized, with pH 7.0 and agitation rate of 350 rpm found to be optimal. In the fed-batch fermentation, 9.3 g/L of 1,2-propanediol was produced after 144 h of cultivation, and the substrate conversion ratio was 0.2 g/g. This study provides an efficient way of 1,2-propanediol production from glycerol via an endogenous pathway of K. pneumoniae.Key points• 1,2-Propanediol was synthesis from glycerol by a tpiA knocked out K. pneumoniae• Overexpression of mgsA, yqhD, or fucO promote 1,2-propanediol production• 9.3 g/L of 1,2-propanediol was produced in fed-batch fermentation.

Ethylene glycol and glycolic acid production by wild-type Escherichia coli.

Ethylene glycol and glycolic acid are bulk chemicals with a broad range of applications. The ethylene glycol and glycolic acid biosynthesis pathways have been produced by microorganisms and used as a biological route for their production. Unlike the methods that use xylose or glucose as carbon sources, xylonic acid was used as a carbon source to produce ethylene glycol and glycolic acid in this study. Amounts of 4.2 g/L of ethylene glycol and 0.7 g/L of glycolic acid were produced by a wild-type Escherichia coli W3110 within 10 H of cultivation with a substrate conversion ratio of 0.5 mol/mol. Furthermore, E. coli strains that produce solely ethylene glycol or glycolic acid were constructed. 10.3 g/L of glycolic acid was produced by E. coli ΔyqhD+aldA, and the achieved conversion ratio was 0.56 mol/mol. Similarly, the E. coli ΔaldA+yqhD produced 8.0 g/L of ethylene glycol with a conversion ratio of 0.71 mol/mol. Ethylene glycol and glycolic acid production by E. coli on xylonic acid as a carbon source provides new information on the biosynthesis pathway of these products and opens a novel way of biomass utilization.

Engineering Bacillus subtilis ATCC 6051a for the production of recombinant catalases.

Catalases are a large group of enzymes that decompose hydrogen peroxide to oxygen and hydrogen, and have been applied widely in numerous areas. Bacillus subtilis ATCC 6051a is a well-known host strain for high level secretion of heterologous peptides. However, the application of 6051a was seriously hampered by insufficient transformation efficiency. In this study, D-xylose inducible comK was integrated into the genome of B. subtilis ATCC 6051a, generating 164S, a mutant owns a transformation efficiency of 1 000-fold higher than its parent strain, thus allowing gene replacement by double crossover recombination using linear dsDNAs. The efficiency of the flanking arms for homologous recombination was then analyzed. We found that 400 bp was the minimal length of homologous fragments required to initiate efficient recombination in the 164S strain. In addition, DNA cassettes encoding two mesophilic catalases (Orf 2-62 and Orf 2-63) from B. licheniformis were integrated onto 164S. The catalytic properties of recombinant Orf 2-62 and Orf 2-63 were analyzed, and were found to be predominantly secreted into the fermentation broth, although they obviously lack any known secretory signal peptide. This work demonstrated that B. subtilis 164S is an excellent cell tool, not only for its superior secretion capacity, but also for its convenience in genetic modification.

Removal of antibiotic resistance genes from swine wastewater by membrane filtration treatment.

Antibiotic resistance genes (ARGs) have attracted extensive attention as an emerging environmental contaminant potentially threatening humans. One of the main emission sources of ARGs is swine wastewater. In this study, integrated membrane filtration including ultrafiltration and two-stage reverse osmosis was conducted for swine wastewater treatment. The abundances of 16 target ARGs, which accounted for 72.64% of the total ARGs in swine wastewater according to metagenomic sequencing, were quantified by quantitative real-time PCR (qPCR) during each stage of the membrane filtration process. The results showed that integrated membrane filtration could reduce more than 99.0% of conventional pollutants and 99.79% of ARGs (from 3.02 × 108 copy numbers/mL to 6.45 × 105 copy numbers/mL). Principal component analysis (PCA) indicated that the removal efficiency of ARGs subtype by membrane filtration did not depend on ARGs type. However, strong correlations were found between ARGs and the wastewater quality indicators TP, SS and EC according to Cooccurrence patterns, indicating that ARG removal was closely associated with insoluble solid particles and soluble ions in swine wastewater. These results showed that membrane filtration could not only remove conventional pollutants such as nitrogen and phosphorus but also reduce the emerging pollutant of ARGs and decrease the risk of ARGs flowing into natural water.

Molecular and Serological Assays for SARS-CoV-2: Insights from Genome and Clinical Characteristics.

BACKGROUND: The ongoing outbreak of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed a challenge for worldwide public health. A reliable laboratory assay is essential both to confirm suspected patients and to exclude patients infected with other respiratory viruses, thereby facilitating the control of global outbreak scenarios. CONTENT: In this review, we focus on the genomic, transmission, and clinical characteristics of SARS-CoV-2, and comprehensively summarize the principles and related details of assays for SARS-CoV-2. We also explore the quality assurance measures for these assays. SUMMARY: SARS-CoV-2 has some unique gene sequences and specific transmission and clinical features that can inform the conduct of molecular and serological assays in many aspects, including the design of primers, the selection of specimens, and testing strategies at different disease stages. Appropriate quality assurance measures for molecular and serological assays are needed to maintain testing proficiency. Because serological assays have the potential to identify later stages of the infection and to confirm highly suspected cases with negative molecular assay results, a combination of these two assays is needed to achieve a reliable capacity to detect SARS-CoV-2.

Improving L-serine formation by Escherichia coli by reduced uptake of produced L-serine.

BACKGROUND: Microbial de novo production of L-serine, which is widely used in a range of cosmetic and pharmaceutical products, has attracted increasing attention due to its environmentally friendly characteristics. Previous pioneering work mainly focused on L-serine anabolism; however, in this study, it was found that L-serine could be reimported through the L-serine uptake system, thus hampering L-serine production. RESULT: To address this challenge, engineering via deletion of four genes, namely, sdaC, cycA, sstT and tdcC, which have been reported to be involved in L-serine uptake in Escherichia coli, was first carried out in the L-serine producer E. coli ES. Additionally, the effects of these genes on L-serine uptake activity and L-serine production were investigated. The data revealed an abnormal phenomenon regarding serine uptake activity. The serine uptake activity of the ΔsdaC mutant was 0.798 nmol min-1 (mg dry weight) -1 after 30 min, decreasing by 23.34% compared to that of the control strain. However, the serine uptake activity of the single sstT, cycA and tdcC mutants increased by 34.29%, 78.29% and 48.03%, respectively, compared to that of the control strain. This finding may be the result of the increased level of sdaC expression in these mutants. In addition, multigene-deletion strains were constructed based on an sdaC knockout mutant. The ΔsdaCΔsstTΔtdcC mutant strain exhibited 0.253 nmol min-1 (mg dry weight) -1L-serine uptake activity and the highest production titer of 445 mg/L in shake flask fermentation, which was more than three-fold the 129 mg/L production observed for the parent. Furthermore, the ΔsdaCΔsstTΔtdcC mutant accumulated 34.8 g/L L-serine with a yield of 32% from glucose in a 5-L fermenter after 36 h. CONCLUSION: The results indicated that reuptake of L-serine impairs its production and that an engineered cell with reduced uptake can address this problem and improve the production of L-serine in E. coli.

Ethylene glycol and glycolic acid production from xylonic acid by Enterobacter cloacae.

BACKGROUND: Biological routes for ethylene glycol production have been developed in recent years by constructing the synthesis pathways in different microorganisms. However, no microorganisms have been reported yet to produce ethylene glycol naturally. RESULTS: Xylonic acid utilizing microorganisms were screened from natural environments, and an Enterobacter cloacae strain was isolated. The major metabolites of this strain were ethylene glycol and glycolic acid. However, the metabolites were switched to 2,3-butanediol, acetoin or acetic acid when this strain was cultured with other carbon sources. The metabolic pathway of ethylene glycol synthesis from xylonic acid in this bacterium was identified. Xylonic acid was converted to 2-dehydro-3-deoxy-D-pentonate catalyzed by D-xylonic acid dehydratase. 2-Dehydro-3-deoxy-D-pentonate was converted to form pyruvate and glycolaldehyde, and this reaction was catalyzed by an aldolase. D-Xylonic acid dehydratase and 2-dehydro-3-deoxy-D-pentonate aldolase were encoded by yjhG and yjhH, respectively. The two genes are part of the same operon and are located adjacent on the chromosome. Besides yjhG and yjhH, this operon contains four other genes. However, individually inactivation of these four genes had no effect on either ethylene glycol or glycolic acid production; both formed from glycolaldehyde. YqhD exhibits ethylene glycol dehydrogenase activity in vitro. However, a low level of ethylene glycol was still synthesized by E. cloacae ΔyqhD. Fermentation parameters for ethylene glycol and glycolic acid production by the E. cloacae strain were optimized, and aerobic cultivation at neutral pH were found to be optimal. In fed batch culture, 34 g/L of ethylene glycol and 13 g/L of glycolic acid were produced in 46 h, with a total conversion ratio of 0.99 mol/mol xylonic acid. CONCLUSIONS: A novel route of xylose biorefinery via xylonic acid as an intermediate has been established.