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.

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.

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.

Dynamic Microstructure Assembly Driven by Lysinibacillus sp. LF-N1 and Penicillium oxalicum DH-1 Inoculants Corresponds to Composting Performance.

The effects of Lysinibacillus sp. LF-N1 and Penicillium oxalicum DH-1 inoculants (LFPO group) on compost succession and the microbial dynamic structure of co-composting wheat straw and cow manure composting were investigated. The inoculants contributed to longer thermophilic stages, higher temperatures (62.8 °C) and lower microbial diversity in the LFPO treatment compared to the control group (CK). Moreover, LFPO inoculation increased the germination index and accelerated organic matter and lignocellulose degradation in the compost. Microbial analysis confirmed that the inoculants effectively altered the microbial communities. The predominant biomarkers for bacteria and fungi in inoculated compost were members of Lysinibacillus and Penicillium, respectively. Functional prediction showed greater lignocellulose degradation and less pathogen accumulation in the LFPO group. The cooccurrence network analysis showed that the network structure in LFPO compost was greatly simplified compared to that in CK. Bacterial cluster A was dominated by Lysinibacillus, and fungal cluster B was represented by Penicillium, which were significantly correlated with temperature and lignocellulose degradation, respectively (p < 0.05). These results demonstrated that the LF-N1 and DH-1 inoculants drove the bacterial and fungal assemblies to induce physicochemical property changes during cocomposting.

Comparative Chloroplast Genome Analysis of Wax Gourd (Benincasa hispida) with Three Benincaseae Species, Revealing Evolutionary Dynamic Patterns and Phylogenetic Implications.

Benincasa hispida (wax gourd) is an important Cucurbitaceae crop, with enormous economic and medicinal importance. Here, we report the de novo assembly and annotation of the complete chloroplast genome of wax gourd with 156,758 bp in total. The quadripartite structure of the chloroplast genome comprises a large single-copy (LSC) region with 86,538 bp and a small single-copy (SSC) region with 18,060 bp, separated by a pair of inverted repeats (IRa and IRb) with 26,080 bp each. Comparison analyses among B. hispida and three other species from Benincaseae presented a significant conversion regarding nucleotide content, genome structure, codon usage, synonymous and non-synonymous substitutions, putative RNA editing sites, microsatellites, and oligonucleotide repeats. The LSC and SSC regions were found to be much more varied than the IR regions through a divergent analysis of the species within Benincaseae. Notable IR contractions and expansions were observed, suggesting a difference in genome size, gene duplication and deletion, and the presence of pseudogenes. Intronic gene sequences, such as trnR-UCU-atpA and atpH-atpI, were observed as highly divergent regions. Two types of phylogenetic analysis based on the complete cp genome and 72 genes suggested sister relationships between B. hispida with the Citrullus, Lagenaria, and Cucumis. Variations and consistency with previous studies regarding phylogenetic relationships are discussed. The cp genome of B. hispida provides valuable genetic information for the detection of molecular markers, research on taxonomic discrepancies, and the inference of the phylogenetic relationships of Cucurbitaceae.

An Analysis of the Possible Migration Routes of Oedaleus decorus asiaticus Bey-Bienko (Orthoptera: Acrididae) from Mongolia to China.

Oedaleus decorus asiaticus (Bey-Bienko) is a destructive pest in grasslands and adjacent farmland in northern China, Mongolia, and other countries in Asia. It has been supposed that this insect pest can migrate a long distance and then induce huge damages, however, the migration mechanism is still unrevealed. The current study uses insect light trap data from Yanqing (Beijing), together with regional meteorological data to determine how air flow contributes to the long-distance migration of O. decorus asiaticus. Our results indicate that sinking airflow is the main factor leading to the insects' forced landing, and the prevailing northwest wind was associated with O. decorus asiaticus taking off in the northwest and moving southward with the airflow from July to September. Meanwhile, the insects have a strong migratory ability, flying along the airflow for several nights. Thus, when the airflow from the northwest met the northward-moving warm current from the southwest, a large number of insects were dropped due to sinking airflow, resulting in a large outbreak. Our simulations suggest that the source of the grasshoppers involved in these outbreaks during early 2000s in northern China probably is in Mongolia, and all evidence indicates that there are two important immigrant routes for O. decorus asiaticus migration from Mongolia to Beijing. These findings improves our understanding of the factors guiding O. decorus asiaticus migration, providing valuable information to reduce outbreaks in China that have origins from outside the country.

Effect of thermophilic microbial agents on nitrogen transformation, nitrogen functional genes, and bacterial communities during bean dregs composting.

This study explored how a thermophilic microbial agent altered nitrogen transformation, nitrogen functional genes, and bacterial communities during bean dregs composting with (T) and without (CK) a thermophilic microbial agent for 15 days. The results showed that the maximum temperature in T reached 73 °C and remained above 70 °C for 8 days, while that in CK was only 65 °C. The pH in T had essentially stabilized on day 7, while that in CK was still increasing. On day 15, the seed germination index (GI) of T (95%) reached maturity (defined by GI ≥ 85%), while the GI of CK was only 36%. The concentrations of total nitrogen, water-soluble nitrogen, ammonia nitrogen, and nitrate nitrogen in T (2.5%, 18.9 g/kg, 8.75 g/kg, and 1.69 g/kg) were all lower than those in CK (3.6%, 28.9 g/kg, 12.75 g/kg, and 6.82 g/kg). During composting, Bacillus played a major role in nitrogen reduction, nitrogen mineralization, denitrification, and the conversion between nitrite and nitrate. Weissella played a major role in nitrogen assimilation. Komagataeibacter and Bacillus played a major role in nitrogen fixation in CK and T, respectively. Nitrification was not observed during composting. The nosZ gene, which converts nitrous oxide to nitrogen, was found only in T. Network analysis suggested that the average number of neighbours in T was 3.30% higher than that in CK and the characteristic path length in T was 14.15% higher than that in CK. Therefore, the thermophilic microbial agents could cause nitrogen loss but promote the maturity of bean dregs, which have great potential application.

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.

Keystone Taxa Lactiplantibacillus and Lacticaseibacillus Directly Improve the Ensiling Performance and Microflora Profile in Co-Ensiling Cabbage Byproduct and Rice Straw.

Ensiling has been widely applied to cope with agricultural solid waste to achieve organic waste valorization and relieve environmental pressure and feedstuff shortage. In this study, co-ensiling of cabbage leaf byproduct and rice straw was performed with inoculation of Lactiplantibacillusplantarum (LP) to investigate the effects of inoculation on ensiling performance and microflora profiles. Compared to the control, LP inoculation preserved more dry matter (DM) content (283.4 versus 270.9 g·kg-1 fresh matter (FM) on day 30), increased lactic acid (LA) content (52.1 versus 35.8 g·kg-1 dry matter on day 15), decreased pH (3.55 versus 3.79 on day 15), and caused accumulation of acetic acid (AA), butyric acid (BA), and ammonia. The investigation showed that LP inoculation modified microflora composition, especially resisting potential pathogens and enriching more lactic acid bacteria (LAB) (p < 0.05). Moreover, Lactiplantibacillus and Lacticaseibacillus were identified as the keystone taxa that influenced physicochemical properties and interactions in microflora. They were also the main functional species that directly restrained undesirable microorganisms (p < 0.05), rather than indirectly working via metabolite inhibition and substrate competition (p > 0.05). The results of this present study improve the understanding of the underlying effect of LP inoculation on improving silage quality and facilitate the bio-transformation of cabbage byproduct and rice straw as animal feed.

Exogenous Probiotics Improve Fermentation Quality, Microflora Phenotypes, and Trophic Modes of Fermented Vegetable Waste for Animal Feed.

The fermentation of leaf vegetable waste to produce animal feed reduces the environmental impact of vegetable production and transforms leaf vegetable waste into a commodity. We investigated the effect of exogenous probiotics and lignocellulose enzymes on the quality and microbial community of fermented feed (FF) produced from cabbage waste. The addition of exogenous probiotics resulted in increased crude protein (CP) content (p < 0.05), better odor (moderate organic acid and ethanol, with low ammonia-N, p < 0.05), and a lower relative abundance (RA) of pathogens (below 0.4%, p < 0.05) in FF, compared to without. With the addition of exogenous probiotics, only Pediococcus and Saccharomyces were enriched and symbiotic in FF; these were the keystone taxa to reduce the abundance of aerobic, form-biofilms, and pathogenic microorganisms, resulting in an efficient anaerobic fermentation system characterized by facultative anaerobic and Gram-positive bacterial communities, and undefined saprotroph fungal communities. Thus, inoculation of vegetable waste fermentation with exogenous probiotics is a promising strategy to enhance the biotransformation of vegetable waste into animal feed.

Synergy in Efficacy of Artemisia sieversiana Crude Extract and Metarhizium anisopliae on Resistant Oedaleus asiaticus.

In order to explore the synergistic control effect of crude extracts of Artemisia sieversiana and Metarhizium anisopliae on Oedaleus asiaticus, we used different doses of M. anisopliae and crude extracts of A. sieversiana singly and in combination, to determine their toxicities to fourth instar O. asiaticus. The results showed that the combination of 10% crude extract of A. sieversiana with 107 and 108 spores/g M. anisopliae concentrations and the combination of 20% crude extract of A. sieversiana with 107 and 108 spores/g M. anisopliae concentrations had significant effects on the mortality, body weight gain, body length gain, growth rate, and overall performance of O. asiaticus than those of the crude extract of A. sieversiana and M. anisopliae alone. Among them, the 20% A. sieversiana crude extract mixed with 108 spores/g M. anisopliae and 10% A. sieversiana crude extract combined with 107 spores/g M. anisopliae, had the best control efficacy. In order to clarify the biochemical mechanism underlying the immune responses of O. asiaticus to the pesticide treatments, we monitored the activities of four enzymes: superoxidase dismutase (SOD), peroxidase (POD), catalase (CAT), and polyphenol oxidase (PPO). The results showed that the activities of three enzymes (SOD, CAT, and PPO) were significantly increased from the treatment with the combination of M. anisopliae mixed with crude extract of A. sieversiana. Interestingly, compared to the crude extract, the combination treatment did not significantly induce the expression of POD enzyme activity, which may be a biochemical factor for increasing the control effect of the combination treatment. Our results showed that the combination treatment had synergistic and antagonistic effects on host mortality, growth, development, and enzyme activities in O. asiaticus.

Exogenous enzyme amendment accelerates maturity and changes microflora succession in horse and wildlife animal manure co-composting.

Composting has been a rational method to dispose of horse or wildlife animal manures, especially in the developed cities with horse clubs and wildlife parks. However, few studies have focused on the mechanism and improvement methods for composting the horse or wildlife animal manures. In this study, we investigated the effect of exogenous compound enzymes on thermophilic composting, which could potentially support the management of horse and wildlife animal manures. With the presence of exogenous enzymes, the duration of high temperature (> 60 °C) was significantly prolonged (p < 0.05), and the germination index was significantly improved (p < 0.05). More-efficient improvement of composting maturity was associated with the addition of that exogenous enzyme that might influence microflora succession and the interaction among microorganic communities, especially fungal, during the composting process. Furthermore, redundancy and canonical correspondence analyses indicated that the C/N ratio, temperature, and germination index were significant variations to influence bacterial communities (p < 0.05). The dominant Flavobacterium, Thermopolyspora, Thermomonospora, and Chaetomium and Saccobolus could play an essential role in carbohydrate and phytotoxin degradation, while Thermobispora and norank_f_Limnochordaceae could lead to temperature rising.

MicroRNA Expression Profile Analysis of Chlamydomonas reinhardtii during Lipid Accumulation Process under Nitrogen Deprivation Stresses.

Lipid accumulation in various microalgae has been found induced by nitrogen deprivation, and it controls many different genes expression. Yet, the underlying molecular mechanisms still remain largely unknown. MicroRNA (miRNAs) play a critical role in post-transcriptional gene regulation. In this study, miRNAs were hypothesized involved in lipid accumulation by nitrogen deprivation. A deep-sequencing platform was used to explore miRNAs-mediated responses induced by nitrogen deprivation in Chlamydomonas reinhardtii. The eukaryotic orthologous groups of proteins (KOG) function in the predicted target genes of miRNA with response to nitrogen deprivation were mainly involved in signal transduction mechanisms, including transcription, lipid transport, and metabolism. A total of 109 miRNA were predicted, including 79 known miRNA and 30 novel miRNA. A total of 29 miRNAs showed significantly differential expressions after nitrogen deprivation, and most of them were upregulated. A total of 10 miRNAs and their targeting genes might involve in lipid transport and metabolism biological process. This study first investigates nitrogen deprivation-regulated miRNAs in microalgae and broadens perspectives on miRNAs importance in microalgae lipid accumulation via nitrogen deprivation. This study provides theoretical guidance for the application of microalgae in bio-oil engineering production.

Inhibitory Effects of Plant Trypsin Inhibitors Msti-94 and Msti-16 on Therioaphis trifolii (Monell) (Homoptera: Aphididae) in Alfalfa.

The spotted alfalfa aphid (Therioaphis trifolii (Monell)) is a known destructive pest that can significantly reduce alfalfa yields. Two differentially up-regulated alfalfa trypsin inhibitors 'Msti-94' and 'Msti-16' in transcriptome were verified in terms of their mRNA levels using RT-qPCR. The prokaryotic expression vector was constructed and its biological functions, including phenotypic and physiological responses, were verified through feeding spotted alfalfa aphids with active recombinant protein mixed with an artificial diet. Gene clone and gene prokaryotic expression confirmed that Msti-94 had a size of 651 bp, encoded 216 amino acids with a predicted protein weight of 23.5 kDa, and a pI value of 6.91. Similarly, the size of Msti-16 was 612 bp, encoded 203 amino acids, and had a predicted protein weight of 22.2 kDa with a pI value of 9.06. We concluded that both Msti-94 and Msti-16 acted as a stomach poison with survival rates reduced to 21.7% and 18.3%, respectively, as compared to the control, where the survival rate was significantly (p < 0.05) higher (60.0%). Aphid reproduction rates were significantly reduced, after 72 h of feeding, in both the Msti-94 and Msti-16 treatments compared to the controls. A concentration of 800 µg/mL (0.8 mg/mL) of recombinant protein and 5000 µg/mL (5 mg/mL) of recombinant expressing bacteria that inhibits the total protease, which ultimately disrupted the activity of trypsin, chymotrypsin, and aminopeptidase.

Large manipulative experiments revealed variations of insect abundance and trophic levels in response to the cumulative effects of sheep grazing.

Livestock grazing can affect insects by altering habitat quality; however, the effects of grazing years and intensities on insect abundance and trophic level during manipulative sheep grazing are not well understood. Therefore, we investigated these effects in a large manipulative experiment from 2014 to 2016 in the eastern Eurasian steppe, China. Insect abundance decreased as sheep grazing intensities increased, with a significant cumulative effect occurring during grazing years. The largest families, Acrididae and Cicadellidae, were susceptible to sheep grazing, but Formicidae was tolerant. Trophic primary and secondary consumer insects were negatively impacted by increased grazing intensities, while secondary consumers were limited by the decreased primary consumers. Poor vegetation conditions caused by heavy sheep grazing were detrimental to the existence of Acrididae, Cicadellidae, primary and secondary consumer insects, but were beneficial to Formicidae. This study revealed variations in insect abundance and trophic level in response to continuous sheep grazing in steppe grasslands. Overall, our results indicate that continuous years of heavy- and over- sheep grazing should be eliminated. Moreover, our findings highlight the importance of more flexible sheep grazing management and will be useful for developing guidelines to optimize livestock production while maintaining species diversity and ecosystem health.