Pararhizobium qamdonense sp. nov., Isolated from an Alpine Soil in Tibet and the Reclassification of Rhizobium gei Shi et al. 2016 as Pararhizobium gei comb. nov.

A novel Gram-stain-negative, aerobic, rod-shaped bacterium named T808T was isolated from an alpine soil in Qamdo, Tibet, PR China. Strain T808T grew at 5-30℃, pH 5.0-9.0 (optimum, 25℃ and pH 7.0-8.0) with 0-2% (w/v) NaCl (optimum, 0%). The 16S rRNA gene sequences of strain T808T showed the highest similarity with Pararhizobium herbae CCBAU83011T (98.8%), followed by Pararhizobium polonicum F5.1T (98.7%), Pararhizobium giardinii H152T (98.5%), Rhizobium gei ZFJT-2 T (98.4%), and Pararhizobium antarcticum NAQVI59T (97.5%). The highest digital DNA-DNA hybridization (dDDH), core-proteome average amino acid identity (cpAAI) and average nucleotide identity (ANI) values between strain T808T and related strains were estimated as 28.0%, 92.1% and 84.4%, respectively. Phylogenetic analysis based on 16S rRNA, core-proteome and whole-genome indicated that strain T808T belonged to the genus Pararhizobium. The genome size was 6.24 Mbp with genomic DNA G + C content of 60.1%. The major cellular fatty acids were Summed feature 8 (C18:1 ω7c or C18:1 ω6c), C16:0 and C19:0 cyclo ω8c. The polar lipids were diphosphatidyl glycerol, phosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidyl choline and unidentified aminophospholipid. The isoprenoid quinone were ubiquinone-10 and ubiquinone-9. Based on phenotypic, phylogenetic, and genotypic data, strain T808T is considered to represent a novel species of the genus Pararhizobium, for which the name Pararhizobium qamdonense sp. nov. is proposed. The type strain is T808T (= JCM 36247 T = CICC 25216 T). According to phylogenetic coherence based on 16S rRNA, core-proteome and whole-genome, it is also proposed that the type strain Rhizobium gei Shi et al. 2016 should be reclassified as Pararhizobium gei comb. nov., the type strain is ZFJT-2 T (= CCTCC AB 2013015 T = KCTC 32301 T = LMG 27603 T).

Neorhizobium xiangyangii sp. nov., isolated from a highland barley cultivation soil in Qamdo, Tibet.

A novel Gram-negative, aerobic, rod-shaped and non-nitrogen fixing bacterium named T786T was isolated from a highland barley cultivation soil in Qamdo, Tibet Autonomous Region, PR China. Strain T786T grew at 5-30 â„ƒ and pH 6.0-10.0 (optimum, 20-25 â„ƒ and pH 7.0-8.0) with 0-4% (w/v) NaCl (optimum, 0%). The 16S rRNA gene sequences of strain T786T showed the highest similarity to Neorhizobium vignae CCBAU 05176T (98.7%), followed by Neorhizobium alkalisoli CCBAU 01393T (98.5%), Neorhizobium tomejilense T17_20T (98.4%), Neorhizobium huautlense S02T (98.4%), and Neorhizobium galegae ATCC 43677T (98.0%). Phylogenetic analysis based on 16S rRNA genes indicated that strain T786T was a new member of the genus Neorhizobium. The digital DNA-DNA hybridization and average nucleotide identity values between strain T786T and related strains were estimated as 20.2-20.6% and 76.6-80.0%, respectively. The genomic DNA G + C content based on the draft genome sequence was 60.2%. The major cellular fatty acids were Summed feature 8 (C18:1 ω7c or C18:1 ω6c), C16:0 and Summed feature 3 (C16:1 ω7c or C16:1 ω6c). The polar lipids were diphosphatidyl glycerol, phosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidyl methyl ethanolamine, unidentified phospholipid and unidentified lipids (1-4). The isoprenoid quinone was ubiquinone-10. The DAP and sugar components of cell wall were meso-DAP and ribose, glucose, respectively. Based on phenotypic, phylogenetic, and genotypic data, for which the name Neorhizobium xiangyangii sp. nov. is proposed. The type strain is T786T (= JCM 35100T = CICC 25102T).

Acinetobacter tibetensis sp. nov., Isolated from a Soil Under a Greenhouse in Tibet.

A Gram-stain-negative, light yellow, aerobic, non-motile, short rod-shaped bacterium named strain Y-23T with iprodione-degrading capability was isolated from a soil under a greenhouse in Tibet, PR China. Strain Y-23T grew at 4-37 â„ƒ and pH 5.0-9.0 (optimum, 25 â„ƒ and pH 7.0) with 0-3% (w/v) NaCl (optimum, 0%). Phylogenetic analysis based on 16S rRNA gene and chromosome genome indicated that strain Y-23T formed a stable evolutionary branch with Acinetobacter tandoii DSM 14970T. The 16S rRNA gene similarity, digital DNA-DNA hybridization and average nucleotide identity values between strain Y-23T and Acinetobacter tandoii DSM 14970T were 98.31%, 43.2% and 91.2%, respectively. The genome size was 3.39 Mbp with a genomic DNA G+C content of 40.59 mol%. The predominant fatty acids were C18:1 ω9c, Summed feature 3 (C16:1 ω7c/C16:1 ω6c), C12:0, C12:0 3-OH and C16:0. The polar lipids were diphosphatidyl glycerol, phosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidyl choline, unidentified phospholipid, four unidentified aminophospholipids and two unidentified lipids. The isoprenoid quinone was Q-8 (19.43%) and Q-9 (80.57%). Based on phenotypic, phylogenetic, and genotypic data, strain Y-23T is considered to represent a novel species of the genus Acinetobacter, for which the name Acinetobacter tibetensis sp. nov. is proposed. The type strain is Y-23T (= CICC 25150T = JCM 35630T).

Improving the Acid Resistance of Tannase TanBLp (AB379685) from Lactobacillus plantarum ATCC14917T by Site-Specific Mutagenesis.

Tannin acyl hydrolase referred commonly as tannase catalyzes the hydrolysis of the galloyl ester bond of tannin to release gallic acid. The tannase TanBLp which cloned from Lactobacillus plantarum ATCC14917T has high activity in the pH range (7.0-9.0) at 40 °C, it would be detrimental to the utilization at acidic environment. The catalytic sites and stability of TanBLp were analyzed using bioinformatics and site-specific mutagenesis. The results reiterated that the amino acid residues Ala164, Lys343, Glu357, Asp421 and His451 had played an important role in maintaining the activity. The optimum pH of mutants V75A, G77A, N94A, A164S and F243A were shifted from 8.0 to 6.0, and mutant V75A has the highest pH stability and activity at acidic conditions than other mutants, which was more suitable for industrial application to manufacture gallic acid. This study was of great significance to promote the industrialization and efficient utilization of tannase TanBLp.

Engineering Yarrowia lipolytica for sustainable Cis-13, 16-docosadienoic acid production.

Cis-13, 16-docosadienoic acid (DDA) is an omega-6 polyunsaturated fatty acid with great potential for application in medicine and health. Using microbial cell factories for DDA production is considered a viable alternative to extracting DDA from plant seeds. In this study, using Yarrowia lipolytica Po1f (Δku70) as a chassis, firstly, the adaptation of three elongases in Po1f (Δku70) were explored. Secondly, the DDA biosynthetic pathway was redesigned, resulting in a DDA content of 0.046 % of total fatty acids (TFAs). Thirdly, through the "push-pull" strategy, the DDA content increased to 0.078 % of TFAs. By enhancing the supply of acetyl-CoA, the DDA production in the engineered strain YL-7 reached 0.391 % of the TFAs (3.19 mg/L). Through optimizing the fermentation conditions, the DDA titer of YL-7 reached 29.34 mg/L. This research achieves the sustainable biological production of DDA in Y. lipolytica.

Structural characterization of extracellular polysaccharides from Phellinus igniarius SH-1 and their therapeutic effects on DSS induced colitis in mice.

Phellinus igniarius is a valuable medicinal and edible mushroom, and its polysaccharides exhibit excellent anti-inflammatory activity. During liquid fermentation to produce P. igniarius mycelia, the fermentation liquid is often discarded, but it contains extracellular polysaccharides. To better utilize these resources, P. igniarius SH-1 was fermented in a 100 L fermenter, and PIPS-2 was isolated and purified from the fermentation broth. The structural characteristics and anti-inflammatory activity of PIPS-2 were determined. PIPS-2 had a molecular weight of 22.855 kDa and was composed of galactose and mannose in a molar ratio of 0.38:0.62. Structural analysis revealed that the main chain of PIPS-2 involved →2)-α-D-Manp-(1 → 3)-ß-D-Galf-(1→, and the side chains involved α-D-Manp-(1 → 6)-α-D-Manp-(1→, α-D-Manp-(1 → 3)-α-D-Manp-(1→, and α-D-Manp-(1. PIPS-2 alleviated the symptoms of dextran sodium sulfate (DSS)-induced colitis in mice, improved the imbalance of inflammatory factors and antioxidant enzymes, and increased short-chain fatty acid contents. Combining the intestinal flora and metabolite results, PIPS-2 was found to regulate the abundance of Firmicutes, Lachnospiraceae_NK4A136_group, Proteobacteria, Bacteroides, and many serum metabolites including hexadecenal, copalic acid, 8-hydroxyeicosatetraenoic acid, artepillin C, and uric acid, thereby ameliorating metabolite related disorders in mice with colitis. In summary, PIPS-2 may improve colitis in mice by regulating the gut microbiota and metabolites.

Metabolic engineering of Yarrowia lipolytica for high-level production of squalene.

Squalene is an important triterpene with a wide range of applications. Given the growing market demand for squalene, the development of microbial cell factories capable of squalene production is considered a sustainable method. This study aimed to investigate the squalene production potential of Yarrowia lipolytica. First, HMG-CoA reductase from Saccharomyces cerevisiae and squalene synthase from Y. lipolytica was co-overexpressed in Y. lipolytica. Second, by enhancing the supply of NADPH in the squalene synthesis pathway, the production of squalene in Y. lipolytica was effectively increased. Furthermore, by constructing an isoprenol utilization pathway and overexpressing YlDGA1, the strain YLSQ9, capable of producing 868.1 mg/L squalene, was obtained. Finally, by optimizing the fermentation conditions, the highest squalene concentration of 1628.2 mg/L (81.0 mg/g DCW) in Y. lipolytica to date was achieved. This study demonstrated the potential for achieving high squalene production using Y. lipolytica.

Increased Cordycepin Production in Yarrowia lipolytica Using Combinatorial Metabolic Engineering Strategies.

As the first nucleoside antibiotic discovered in fungi, cordycepin, with its various biological activities, has wide applications. At present, cordycepin is mainly obtained from the natural fruiting bodies of Cordyceps militaris. However, due to long production periods, low yields, and low extraction efficiency, harvesting cordycepin from natural C. militaris is not ideal, making it difficult to meet market demands. In this study, an engineered Yarrowia lipolytica YlCor-18 strain, constructed by combining metabolic engineering strategies, achieved efficient de novo cordycepin production from glucose. First, the cordycepin biosynthetic pathway derived from C. militaris was introduced into Y. lipolytica. Furthermore, metabolic engineering strategies including promoter, protein, adenosine triphosphate, and precursor engineering were combined to enhance the synthetic ability of engineered strains of cordycepin. Fermentation conditions were also optimized, after which, the production titer and yields of cordycepin in the engineered strain YlCor-18 under fed-batch fermentation were improved to 4362.54 mg/L and 213.85 mg/g, respectively, after 168 h. This study demonstrates the potential of Y. lipolytica as a cell factory for cordycepin synthesis, which will serve as the model for the green biomanufacturing of other nucleoside antibiotics using artificial cell factories.

Reprogramming the fatty acid metabolism of Yarrowia lipolytica to produce the customized omega-6 polyunsaturated fatty acids.

Omega-6 polyunsaturated fatty acids (ω6-PUFAs), such as γ-linolenic acid (GLA), dihomo-γ-linolenic acid (DGLA) and arachidonic acid (ARA), are indispensable nutrients for human health. Harnessing the lipogenesis pathway of Yarrowia lipolytica creates a potential platform for producing customized ω6-PUFAs. This study explored the optimal biosynthetic pathways for customized production of ω6-PUFAs in Y. lipolytica via either the Δ6 pathway from Mortierella alpina or the Δ8 pathway from Isochrysis galbana. Subsequently, the proportion of ω6-PUFAs in total fatty acids (TFAs) was effectively increased by bolstering the provision of precursors for fatty acid biosynthesis and carriers for fatty acid desaturation, as well as preventing fatty acid degradation. Finally, the proportions of GLA, DGLA and ARA synthesized by customized strains accounted for 22.58%, 46.65% and 11.30% of TFAs, and the corresponding titers reached 386.59, 832.00 and 191.76 mg/L in shake-flask fermentation, respectively. This work provides valuable insights into the production of functional ω6-PUFAs.

Efficient production of cordycepin by engineered Yarrowia lipolytica from agro-industrial residues.

Cordycepin, a nucleoside compound with a variety of biological activities, has been extensively applied in the nutraceutical and pharmaceutical industries. The advancement of microbial cell factories using agro-industrial residues provides a sustainable pathway for cordycepin biosynthesis. Herein, the cordycepin production was enhanced by the modification of glycolysis and pentose phosphate pathway in engineered Yarrowia lipolytica. Then, cordycepin production based on economical and renewable substrates (sugarcane molasses, waste spent yeast, and diammonium hydrogen phosphate) was analyzed. Furthermore, the effects of C/N molar ratio and initial pH on cordycepin production were evaluated. Results indicated that the maximum cordycepin productivity of 656.27 mg/L/d (72 h) and cordycepin titer was 2286.04 mg/L (120 h) by engineered Y. lipolytica in the optimized medium, respectively. The cordycepin productivity in the optimized medium was increased by 28.81% compared with the original medium. This research establishes a promising way for efficient cordycepin production from agro-industrial residues.

Effects of different boiling processes on chemical compositions of Lilii Bulbus soup.

Lilii Bulbus, an edible Chinese herbal medicine, has a long history in medicine. However, research on effectively boiling Lilii Bulbus is rare. To make the more nutritious Lilii Bulbus soup, the optimized boiling process, using an alternate heating mode by decoction pot carrying a mixture of water and Chinese liquor at the ration of 9:1, was established in this study. Compared to the soup prepared by the daily process, the polysaccharide amount improved by 54%, and the total heavy metals decreased by 33.5% using the optimized boiling process. In addition, the total saponins at 34.3 µg/g were determined in the soup prepared by the optimized process. Meanwhile, the colchicine content in the boiled Lilii Bulbus soup was undetectable using the optimized process. This research performs an optimized boiling process for making Lilii Bulbus soup, and provides a reference for generating high commercial value from Lilii Bulbus soup in the future.

Degradation of iprodione by a novel strain Azospirillum sp. A1-3 isolated from Tibet.

A bacterial strain A1-3 with iprodione-degrading capabilities was isolated from the soil for vegetable growing under greenhouses at Lhasa, Tibet. Based on phenotypic, phylogenetic, and genotypic data, strain A1-3 was considered to represent a novel species of genus Azospirillum. It was able to use iprodione as the sole source of carbon and energy for growth, 27.96 mg/L (50.80%) iprodione was reduced within 108 h at 25°C. During the degradation of iprodione by Azospirillum sp. A1-3, iprodione was firstly degraded to N-(3,5-dichlorophenyl)-2,4-dioxoimidazolidine, and then to (3,5-dichlorophenylurea) acetic acid. However, (3,5-dichlorophenylurea) acetic acid cannot be degraded to 3,5-dichloroaniline by Azospirillum sp. A1-3. A ipaH gene which has a highly similarity (98.72-99.92%) with other previously reported ipaH genes, was presented in Azospirillum sp. A1-3. Azospirillum novel strain with the ability of iprodione degradation associated with nitrogen fixation has never been reported to date, and Azospirillum sp. A1-3 might be a promising candidate for application in the bioremediation of iprodione-contaminated environments.

High-level de novo biosynthesis of cordycepin by systems metabolic engineering in Yarrowia lipolytica.

Cordycepin is a nucleoside antibiotic with various biological activities, which has wide applications in the area of cosmetic and medicine industries. However, the current production of cordycepin is costly and time-consuming. To construct the promising cell factory for high-level cordycepin production, firstly, the design and construction of cordycepin biosynthetic pathway were performed in Yarrowia lipolytica. Secondly, the adaptivity between cordycepin biosynthetic pathway and Y. lipolytica was enhanced by enzyme fusion and integration site engineering. Then, the production of cordycepin was improved by the enhancement of adenosine supply. Furthermore, through modular engineering, the production of cordycepin was achieved at 3588.59 mg/L from glucose. Finally, 3249.58 mg/L cordycepin with a yield of 76.46 mg/g total sugar was produced by the engineered strain from the mixtures of glucose and molasses. This research is the first report on the de novo high-level production of cordycepin in the engineered Y. lipolytica.

[Advances in the biosynthesis of pentostatin].

Pentostatin is a nucleoside antibiotics with a strong inhibitory effect on adenosine deaminase, and is widely used in the clinical treatment of malignant tumors. However, the high cost hampers its application. In the past 10 years, the biosynthesis of pentostatin were focused on strain breeding, optimization of medium composition and fermentation process. To date, there are no reviews summarizing the elucidated biosynthetic mechanism of pentostatin. This review starts by introducing the various chemical route for production of pentostatin, followed by summarizing the mechanisms of pentostatin biosynthesis in different microorganisms. Finally, challenges for biosynthesis of pentostatin were discussed, and strategies for regulating and improving the microbial synthesis of pentostatin were proposed.

Rhamnolipid Enhances the Nitrogen Fixation Activity of Azotobacter chroococcum by Influencing Lysine Succinylation.

The enhancement of nitrogen fixation activity of diazotrophs is essential for safe crop production. Lysine succinylation (KSuc) is widely present in eukaryotes and prokaryotes and regulates various biological process. However, knowledge of the extent of KSuc in nitrogen fixation of Azotobacter chroococcum is scarce. In this study, we found that 250 mg/l of rhamnolipid (RL) significantly increased the nitrogen fixation activity of A. chroococcum by 39%, as compared with the control. Real-time quantitative reverse transcription PCR (qRT-PCR) confirmed that RL could remarkably increase the transcript levels of nifA and nifHDK genes. In addition, a global KSuc of A. chroococcum was profiled using a 4D label-free quantitative proteomic approach. In total, 5,008 KSuc sites were identified on 1,376 succinylated proteins. Bioinformatics analysis showed that the addition of RL influence on the KSuc level, and the succinylated proteins were involved in various metabolic processes, particularly enriched in oxidative phosphorylation, tricarboxylic acid cycle (TCA) cycle, and nitrogen metabolism. Meanwhile, multiple succinylation sites on MoFe protein (NifDK) may influence nitrogenase activity. These results would provide an experimental basis for the regulation of biological nitrogen fixation with KSuc and shed new light on the mechanistic study of nitrogen fixation.

Metabolic engineering of Yarrowia lipolytica for improving squalene production.

The aim of this present research is to enhance the squalene production in Yarrowia lipolytica using pathway engineering and bioprocess engineering. Firstly, to improve the production of squalene, the endogenous HMG-CoA reductase (HMG1) was overexpressed in Y. lipolytica to yield 208.88 mg/L squalene. Secondly, the HMG1 and diacylglycerol acyltranferase (DGA1) were co-overexpressed, the derived recombinant Y. lipolytica SQ-1 strain produced 439.14 mg/L of squalene. Thirdly, by optimizing the fermentation medium, the improved titer of squalene with 514.34 mg/L was obtained by the engineered strain SQ-1 grown on YPD-80 medium. Finally, by optimizing the addition concentrations of acetate, citrate and terbinafine, the 731.18 mg/L squalene was produced in the engineered strain SQ-1 with the addition of 0.5 mg/L terbinafine. This work describes the highest reported squalene titer in Y. lipolytica to date. This study will provide the foundation for further engineering Y. lipolytica capable of cost-efficiently producing squalene.

Diversity of Culturable Bacteria Isolated from Highland Barley Cultivation Soil in Qamdo, Tibet Autonomous Region.

The soil bacterial communities have been widely investigated. However, there has been little study of the bacteria in Qinghai-Tibet Plateau, especially about the culturable bacteria in highland barley cultivation soil. Here, a total of 830 individual strains were obtained at 4°C and 25°C from a highland barley cultivation soil in Qamdo, Tibet Autonomous Region, using fifteen kinds of media. Seventy-seven species were obtained, which belonged to 42 genera and four phyla; the predominant phylum was Actinobacteria (68.82%), followed by Proteobacteria (15.59%), Firmicutes (14.29%), and Bacteroidetes (1.30%). The predominant genus was Streptomyces (22.08%, 17 species), followed by Bacillus (6.49%, five species), Micromonospora (5.19%, four species), Microbacterium (5.19%, four species), and Kribbella (3.90%, three species). The most diverse isolates belonged to a high G+C Gram-positive group; in particular, the Streptomyces genus is a dominant genus in the high G+C Gram-positive group. There were 62 species and 33 genera bacteria isolated at 25°C (80.52%), 23 species, and 18 genera bacteria isolated at 4°C (29.87%). Meanwhile, only eight species and six genera bacteria could be isolated at 25°C and 4°C. Of the 77 species, six isolates related to six genera might be novel taxa. The results showed abundant bacterial species diversity in the soil sample from the Qamdo, Tibet Autonomous Region.

Yarrowia lipolytica as an Oleaginous Platform for the Production of Value-Added Fatty Acid-Based Bioproducts.

The microbial fermentation process has been used as an alternative pathway to the production of value-added natural products. Of the microorganisms, Yarrowia lipolytica, as an oleaginous platform, is able to produce fatty acid-derived biofuels and biochemicals. Nowadays, there are growing progresses on the production of value-added fatty acid-based bioproducts in Y. lipolytica. However, there are fewer reviews performing the metabolic engineering strategies and summarizing the current production of fatty acid-based bioproducts in Y. lipolytica. To this end, we briefly provide the fatty acid metabolism, including fatty acid biosynthesis, transportation, and degradation. Then, we introduce the various metabolic engineering strategies for increasing bioproduct accumulation in Y. lipolytica. Further, the advanced progress in the production of fatty acid-based bioproducts by Y. lipolytica, including nutraceuticals, biofuels, and biochemicals, is summarized. This review will provide attractive thoughts for researchers working in the field of Y. lipolytica.

Improved Production of Arachidonic Acid by Combined Pathway Engineering and Synthetic Enzyme Fusion in Yarrowia lipolytica.

Arachidonic acid (ARA, C20:4) is a typical ω-6 polyunsaturated fatty acid with special functions. Using Yarrowia lipolytica as an unconventional chassis, we previously showed the performance of the Δ-6 pathway in ARA production. However, a significant increase in the Δ-9 pathway has rarely been reported. Herein, the Δ-9 pathway from Isochrysis galbana was constructed via pathway engineering, allowing us to synthesize ARA at 91.5 mg L-1. To further improve the ARA titer, novel enzyme fusions of Δ-9 elongase and Δ-8 desaturase were redesigned in special combinations containing different linkers. Finally, with the integrated pathway engineering and synthetic enzyme fusion, a 29% increase in the ARA titer, up to 118.1 mg/L, was achieved using the reconstructed strain RH-4 that harbors the rigid linker (GGGGS). The results show that the combined pathway and protein engineering can significantly facilitate applications of Y. lipolytica.

Biotechnological applications of Yarrowia lipolytica: Past, present and future.

Non-conventional yeasts have attracted increasing interest due to their biochemical characteristics and potential applications. Yarrowia lipolytica is a non-conventional yeast with specific characteristics and physiology. The potential physiological and metabolic capabilities of Y. lipolytica, which can assimilate many different carbon sources, including typical hydrophilic and hydrophobic materials, are reviewed in this paper. Concerning the uptake and metabolism substrates, this review focuses particularly on low-cost raw materials, such as glycerol. Moreover, this review presents the results of safety studies of Y. lipolytica. Finally, the wide applications of Y. lipolytica, such as functional enzyme production, metabolite synthesis and environmental bioremediation, are reviewed in this paper. Recently, with the development of system biology and synthetic biology, it was concluded that these technologies will provide new opportunities for potential applications of Y. lipolytica in the future.