分枝杆菌LY-1内源性表达元件的筛选及其在降低Cas9蛋白毒性中的应用

【背景】分枝杆菌LY-1因能够将天然植物甾醇代谢转化为重要甾体药物中间体,目前已成为工业上的优势生产菌株。高效的CRISPR/Cas9基因编辑技术是工业菌株代谢工程改造进行产量性状提升的关键。然而由于Cas9蛋白的高表达毒性问题且分枝杆菌中已公开报道的可用表达元件较少,极大地限制了Cas9蛋白在该菌株中的适度表达。【目的】筛选内源性表达元件,利用合适的表达元件启动Cas9蛋白的表达,降低其对菌株的毒性。【方法】依据文献和前期研究获得的分枝杆菌基因转录组水平数据,并结合启动子在线预测网站BDGP(https://www.fruitfly.org/seq＿tools/promoter.html),筛选内源性表达元件。以增强型绿色荧光蛋白作为报告基因对表达元件的强度进行评估,并采用不同强度的表达元件启动Cas9蛋白的表达。【结果】获得了23个不同表达强度的表达元件,采用中等强度的表达元件及弱表达元件都降低了Cas9蛋白对分枝杆菌LY-1的毒性,实现了Cas9蛋白在该菌株中的适度表达。【结论】建立了分枝杆菌LY-1内源性表达元件库,为后续菌株中高效CRISPR/Cas9基因编辑技术的构建及关键...     

Luffa cylindrica and phytosterols bioconversion: from shake flask to jar bioreactor

Bioconversion of lipophilic compounds poorly soluble in water, such as sterols, required the use of chemicals and solubilizing agents. On the other hand, it was shown that immobilization of Mycobacterium species on the dried fruit of Luffa cylindrica (DFLC) allows a close interaction between immobilized cells and cholesterol particles and increases by then the product’s yield. In this work, the use of DFLC in a 5-l jar bioreactor with phytosterols mixture (1 g/l) as substrate was assessed without addition of any chemicals or solubilizing agents. DFLC increased by a factor of four the volumetric productivity of androstenones (0.08 g/l day). Products were accumulated in the aqueous medium while substrates remained on the fibers of DFLC. This observation lets envisage a green semi-continuous process of androstenone production. DFLC has no influence on cell growth, and is moreover natural, inexpensive, non-toxic, and mechanically strong.     

Dependence of steroid 1(2)-dehydrogenation on the C-17 side chain during cholesterol metabolism by immobilized Mycobacterium fortuitum

1(2)-Dehydrogenation of 4-cholestene-3-one by immobilized Mycobacterium fortuitum NRRL B-8153 and free growing Mycobacterium sp. NRRL B-3805 and Micromonospora chlacea MTCC 329 depended on the nature of the C-17 side chain. The 1(2)-dehydrogenation did not appear to occur before C-17-side-chain cleavage of the 4-cholestene-3-one.S. Patil is and A. Srivastava was with the School of Life Sciences, Devi Ahilya Vishwavidyalaya. Vigyan Bhawan, Khandwa Road Campus, Indore-452 001, India. A. Srivastava is now with the Shripati Singhania R&D Centre. J. K. Pharmaceuticals. 13th Mile Stone. Mathura Road, Faridabad (Haryana) 121 003, India.     

Identification of metabolites from degradation of naphthalene by a Mycobacterium sp.

A Mycobacterium sp. isolated from oil-contaminated sediments was previously shown to mineralize 55% of the added naphthalene to carbon dioxide after 7 days of incubation. In this paper, we report the initial steps of the degradation of naphthalene by a Mycobacterium sp. as determined by isolation of metabolites and incorporation of oxygen from ¹⁸O₂ into the metabolites. The results indicate that naphthalene is initially converted to cis- and trans-1,2-dihydroxy-1,2-dihydronaphthalene by dioxygenase and monooxygenase catalyzed reactions, respectively. The ratio of the cis to trans-naphthalene dihydrodiol isomers was approximately 25:1. Thin layer and high pressure liquid chromatographic and mass spectrometric techniques indicated that besides the cis- and trans-1,2-dihydroxy-1,2-dihydronaphthalene, minor amounts of ring cleavage products salicylate and catechol were also formed. Thus the formation of both cis and trans-naphthalene dihydrodiols by the Mycobacterium sp. is unique. The down-stream reactions to ring cleavage products proceed through analogous dioxygenase reactions previously reported for the bacterial degradation of naphthalene.     

Degradation of 2-chlorotoluene by <Emphasis Type="Italic">Rhodococcus</Emphasis> sp. OCT 10

A strain Rhodococcus sp. OCT 10 DSM 45596^T, exhibiting 99.9% of 16S rDNA identity with Rhodococcus wratislaviensis NCIMB 13082, was isolated from a soil sample. The strain completely mineralised 2-chlorotoluene, 2-bromotoluene, o-xylene, benzyl alcohol and benzoate. In contrast, 2-fluorotoluene was only partially mineralised. By GC-MS and ¹H-NMR analyses, 4-chloro-3-methylcatechol was identified as the central intermediate in the degradation pathway of 2-chlorotoluene. It was further degraded by enzymes of the meta cleavage pathway. Catechol 1,2-dioxygenase and chlorocatechol 1,2-dioxygenase as the initial enzymes of the ortho cleavage pathways were not detectable under these conditions. Furthermore, neither formation nor oxidation of 2-chlorobenzylic alcohol, 2-chlorobenzaldehyde, or 2-chlorobenzoate was observed, thereby excluding side chain oxidation activity.     

Vegetable sponge: A new immobilization medium for plant cells

Summary Immobilization of Saccharum officinarum cells within the vegetable sponge of Luffa cylindrica is reported as a new medium for the cells of higher plants. The use of biostructure of the sponge offers an alternative matrix to the ones used presently for immobilization.     

Preparation of androsta-1,4-diene-3,17-dione from sterols using <Emphasis Type="Italic">Mycobacterium neoaurum</Emphasis> VKPM Ac-1656 strain

A product of microbiological cleavage of the sterols side chain, androsta-1,4-diene-3,17-dione, is toxic for bacteria, in particular, actinobacteria of the genera Mycobacterium and Arthrobacter. Sterols were transformed into androsta-1,4-diene-3,17-dione by culturing the M. neoaurum VKPM Ac-1656 strain in a high yield, provided that a sorbent was used for elimination of contact between the bacterial cells and the product. Unlike the cholesterol side chain, the more branched chains of phytosterols were cleaved in the presence of M. neoaurum at a high rate only under turbulent stirring of the culture medium, which intensified the formation of hydrocarbonate ion from NaNI₃ in situ.     

Production of androsta-1,4-diene-3,17-dione from cholesterol using immobilized growing cells of Mycobacterium sp. NRRL B-3683 adsorbed on solid carriers

Summary Living cells of Mycobacterium sp. NRRL B-3683 were immobilized by adsorption on different types of solid carriers in order to produce androsta-1,4-diene-3,17-dione (ADD) from cholesterol. Activated alumina proved to be the most preferred carrier for long-term operation when glucose and peptone were added to the reaction medium. In a repeated-batch process, the maximum productivity of ADD was about 0.19 g/l per day with a molar conversion rate of 77% when 1.0 g/l of cholesterol was added to the reaction medium. The half-life of the immobilized cells was more than 45 days and the system could be reactivated by incubating the immobilized cells in a cell growth medium.     

Immobilization ofColeus blumei cells in a column reactor using a spray feeding system

Summary Coleus blumei cells were immobilized in a column reactor packed withLuffa cylindrica pieces. Medium was fed from the top of the column using a spray system and cells maintained high viability for 52 days. Cell growth was slower but rosmarinic acid production was better compared to immobilized cells in the shake flasks.     

Naturally Occurring Regenerative Differentiation of Xylem Around Adventitious Roots in Luffa cylindrica Seedlings

Regeneration of xylem induced by adventitious root formationin the hypocotyl of Luffa cylindrica Roem. seedlings is described.This naturally occurring form of xylem regeneration involvesthe formation of a bypass of regenerated tracheary elementsaround a root without external severance of the vascular strands.The regeneration of xylem around an adventitious root is polarand is very similar in its developmental pattern to the well-knownxylem regeneration induced by wounding vascular strands. Adventitious root formation, Luffa cylindrica Roem, regenerated tracheary elements, vascular differentiation, xylem regeneration     

Transformation of C19-steroids and testosterone production by sterol-transforming strains of Mycobacterium spp.

Redox conversions of exogenic C₁₉-steroids (androstenedione (AD), androstadienedione (ADD), testosterone and 1(2)-dehydrotestosterone) were studied using a mutant strain of Mycobacterium sp. Et1 with high level of 17β-hydroxysteroid dehydrogenase (17-HSD) activity. Factors affecting target 17β-reduction and side reactions by resting and growing cells were estimated. The effects of glucose supplementation, pH, mode of substrate addition were identified. The results confirmed that double reduction of androstadienedione, both of 17-keto group and 1(2)-double bond, is more effective for testosterone formation than a single reduction of 17-keto group of AD. These findings argued for the application of the strain capable of sterol side chain degradation and expressed 17-HSD, 3-ketosteroid-1(2)-dehydrogenase and 1-ene-reductase activities, for testosterone obtaining from sitosterol. Under optimal conditions, the conversion of sitosterol (5 g/l) by Mycobacterium sp. VKM Ac-1816D in laboratory fermenter resulted in 50–55% molar yield of testosterone.     

Autotoxic potential of cucurbit crops

Soil sickness is often observed in cucurbit crops such as Citrullus lanatus, Cucumis melo and Cucumis sativus, but not in cucurbit crops such as Cucurbita moschata, Lagenaria leucantha and Luffa cylindrica. Results showed that root aqueous extracts of Citrullus lanatus, Cucumis melo and Cucumis sativus were autotoxic, but those of Cucurbita moschata, Momordica charantia and Luffa cylindrica were less autotoxic to the radicle elongation of respective species. Plant growth of Citrullus lanatus, Cucumis melo and Cucumis sativus were greatly inhibited by autotoxic substances released from powered root tissue at a rate of 1 g per seedling. Root exudates of Citrullus lanatus, Cucumis melo and Cucumis sativus were autotoxic to radicle elongation and seedling growth of respective species. However, root exudates of Citrullus lanatus did not inhibit radicle elongation of Cucurbita ficifolia, which is commonly used as rootstock for the grafting of Citrullus lanatus, Cucumis melo and Cucumis sativus to decrease soil-borne diseases in commercial production. It seems possible to overcome autotoxicity in cucurbit crops by grafting on Cucurbita ficifolia. This revised version was published online in June 2006 with corrections to the Cover Date.     

Microbial Transformation of Sterols

Cholesterol decomposing ability of 1589 microbial strains was examined. Two hundreds and thirty six strains from actinomycetes, bacteria, molds, and yeasts were found capable of oxidizing cholesterol into cholestenone. Cholesta-1,4-dien-3-one was produced by 5 strains of Streptomyces. The complete decomposition of cholesterol molecule was observed in the genera: Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Mycobacterium, Nocardia, Protaminobacter, Serratia, and Streptomyces. α,α′-Dipyridyl and arsenite inhibited decomposing enzymes giving rise to cholestenone, cholesta-1,4-dien-3-one, and an intermediate probably devoid of the sterol side chain.

Selective cleavage of the side chains of various sterols at C-17, giving rise to androsta-1,4-diene-3,17-dione (ADD), occurred in the presence of α,α′-dipyridyl by microorganisms of the following genera: Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Mycobacterium, Nocardia, Protaminobacter, Serratia, and Streptomyces. The degradation pathway of cholesterol, for example, was shown as follows:

Other sterols such as campesterol, β-sitosterol, stigmasterol and 7-dehydrocholesterol were degraded by the same sequence. The pathway exemplified in cholesterol is considered to be the general degradation pathway of sterols by their decomposing microorganisms.

It was further demonstrated that ADD thus formed from sterols was converted into 3-hydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione.     

Production and characteristics of the exocellular polysaccharide of a mutantMycobacterium strain

Mutant strains ofMycobacterium sp. V-649 producing highly mucous colonies on a solid cultivation medium were prepared after treatment with N-methyl-N′-nitro-N-nitrosoguanidine and production of the exocellular polysaccharide was tested. The strains were cultivated in media with suitable sugar sources under submerged conditions. It was found thatMycobacterium sp. V649/15 produces a maximum of 15–19% polymer after a 5–6-d cultivation. Gas chromatography indicated that the exocellular polysaccharide produced by this strain is of glucan type.     

<Emphasis Type="Italic">Blighia unijugata</Emphasis> and <Emphasis Type="Italic">Luffa cylindrica</Emphasis> Seed Oils: Renewable Sources of Energy for Sustainable Development in Rural Africa

Self-sufficiency in energy requirement is critical to the success of any developing economy. Apart from the search for alternatives, there is a need to achieve energy independence, directing much focus on biofuels. Biodiesel is simple to use, biodegradable, nontoxic, and essentially free of sulfur and aromatics. Oil was extracted from the seeds of Blighia unijugata and Luffa cylindrica, subjected to chemical characterization and biodiesel production. The oil yield from the seed of B. unijugata was 50.82 ± 1.20% while that of L. cylindrica was 39.10 ± 0.20%. The biodiesel produced had ester content above 98%. The flash point of the biodiesel from B. unijugata and L. cylindrica was above 120°C while the phosphorus content was also below 1 ppm in both cases. The oxidative stability of B. unijugata was 44.30 ± 0.30 h, while that of L. cylindrica was lower than this value due to its high unsaturation. The copper strip corrosion value of the biodiesel was also found to be 1A. This study showed that the high free fatty acid content of B. unijugata and L. cylindrica seed oil can be reduced in a one-step pretreatment of esterification reaction using H₂SO₄ as catalyst thus reducing the problem of soap formation encountered when using oil with high free fatty acid for the production of biodiesel.     

Population Dynamics of Two Toluene Degrading Bacterial Species in a Contaminated Stream

Toluene uptake by a benthic biofilm community was previously shown to vary seasonally from 0.03 m hr⁻¹ in winter to 0.2 m hr⁻¹ in summer in a solvent-contaminated stream of the Aberjona watershed. We used quantitative PCR to estimate the population dynamics of previously isolated species of toluene-degrading Xanthobacter autotrophicus and Mycobacterium sp. in both toluene-contaminated and uncontaminated reaches of the stream, and to estimate their relative roles in overall biodegradation rate. Quantification using specific 16S rDNA primers for X. autotrophicus and Mycobacterium sp. showed that populations of both species were much larger in the toluene-contaminated than the toluene-free reach, in agreement with earlier culture-based investigations. A relatively brief bloom of X. autotrophicus occurred in the contaminated reach in the summer, while Mycobacterium sp. populations occurred at elevated densities for more than 5 months. Calculations showed that Mycobacterium, previously thought to be less important than Xanthobacter in annual toluene degradation based on single time-point CFU estimates, appears actually more important because of this longer persistence.     

Variations in the Bioactive Compounds Composition and Biological Activities of Loofah (Luffa cylindrica) Fruits in Relation to Maturation Stages

The present work aimed to determine the antioxidant and antiproliferative potential of Luffa cylindrica fruits collected at two different maturation stages and to identify and compare their functional components composition. The MeOH extracts of L. cylindrica fruits harvested at 60 – 65 days after seeding (S1) and 85 – 90 days after seeding (S2) were investigated for their antioxidant activity using various assays. Furthermore, the antiproliferative activity of the extracts against HeLa human cervical cancer cells was explored with xCELLigence real time cell analyzer, while the effect of the samples on the membrane integrity of the same cell line was assessed using LDH cytotoxicity leakage assay. Ultimately, the phytochemicals were analyzed using GC/MS and HPLC/TOF‐MS. The S1 sample had higher contents and more diversity in the phenolic compounds composition than S2. Furthermore, the S1 extract showed the highest antioxidant and antiproliferative activity, while the S2 extract had higher cytotoxicity towards HeLa cells. The findings revealed that the time of harvest has a big impact on the phytochemicals content and activity and that harvesting L. cylindrica at an early stage before the beginning of the development of the cellulose fibrous system is recommended for a rich phytochemical composition and efficient antioxidant and antiproliferative activities.     

Genotypic characteristics of a Mycobacterium sp. isolated from yellowtail Seriola quinqueradiata and striped jack Pseudocaranx dentex in Japan

In Japan, a Mycobacterium marinum‐like mycobacterium was isolated from the yellowtail, Seriola quinqueradiata. The species was identified as M. marinum by a commercial mycobacterial DNA‐DNA hybridization kit. Nevertheless, PCR restriction analysis of the DNA of its RNA polymerase β‐subunit gene definitively showed that this Mycobacterium sp. was M. ulcerans. PCR analysis revealed the genotypic characteristics of M. ulcerans in the Mycobacterium sp., only the mup053 gene sequence being absent, as has been found previously in other piscine mycobacteria such as M. marinum strains DL240490 and DL045 and M. pseudoshottsii. With one exception, this Mycobacterium sp. and M. pseudoshottsii had identical 16S rRNA gene sequences, which is also probably true of M. marinum strains DL240490 and DL045. Similarly, according to comparisons of the 16S rRNA gene, ITS region, and hsp65 gene sequences, this Mycobacterium sp. is more closely related to M. pseudoshottsii than to M. ulcerans or M. marinum. A PCR product of approximately 2000 bp was amplified from region of difference 9 in the Mycobacterium sp. The nucleotide sequence revealed insertion of IS2404, the sequence of which is 1366 bp long. The novel single nucleotide polymorphisms identified in this region distinguished this Mycobacterium sp. from M. marinum strain DL240490 and M. pseudoshottsii. The present findings raise the possibility that these species have a common ancestor. Further studies are required to improve our understanding of the relationship between their geographical origin and genetic diversity.     

Herbivory-induced overcompensation and resource-dependent production of extrafloral nectaries in Luffa cylindrica (Cucurbitaceae)

Extrafloral nectaries (EFNs) are nectar secretory structures involved in the indirect defense of plants. In the sponge gourd (Luffa cylindrica), EFNs commonly occur on the lower surface of leaf blades and stipules and remain functional until leaf senescence. To test the hypothesis that the development of EFNs is influenced by herbivore damage and resource availability, we grew Luffa cylindrica under different concentrations of Hoagland's nutrient solution (nutrient-poor conditions: 10%, 50%; and control condition: 100%) and two herbivory treatments (damaged and undamaged leaves). We collected ten leaves from treated plants to quantify leaf area and EFN density. Overall, leaf area increased and EFN decreased in damaged plants, but this significantly depended on nutritional status. In undamaged plants, EFN density tended to remain constant, whereas foliar area increased with nutrient input. Under herbivory, foliar area increased at 10% but decreased at 50 and 100% of nutrients in relation to undamaged plants, whereas EFN density tended to increase with nutrient availability to exceed undamaged plants under control concentrations. Plants under nutrient-poor conditions subjected to herbivory exhibited an increased foliar area, characterizing a compensatory mechanism. Our results suggest that herbivore-induced indirect defense is a damage- and resource-dependent response in Luffa cylindrica. These findings contribute to understanding the factors that modulate indirect defenses and plant-herbivore-environment interactions.     

In situ 1H NMR study of the biodegradation of xenobiotics: application to heterocyclic compounds

In vivo or in situ nuclear magnetic resonance (NMR) offers a powerful tool to study the degradation of xenobiotics by microorganisms. Most studies reported are based on the use of heteronuclei, and experiments with xenobiotics have been limited because specifically labeled xenobiotics are not commercially available, with the exception of ¹⁹F and ³¹P. ¹H NMR is, thus, of great interest in this area. To avoid problems caused by the presence of water and intrinsic metabolite signals, some studies were performed using a deuterated medium or specific detection of protons linked to the ¹³C–¹⁵N enriched pattern. We report here the application of in situ ¹H NMR, performed directly on culture media, to study the metabolism of heterocyclic compounds. In this review, we show that a common pathway is involved in the biodegradation of morpholine, piperidine, and thiomorpholine by Mycobacterium aurum MO1 and Mycobacterium sp. RP1. In all cases, the first step is the cleavage of the C–N bond, which results in an amino acid. Thiomorpholine is first oxidized to sulfoxide before the opening of the ring. The second step is the deamination of the intermediate amino acid, which leads to the formation of a diacid. We have shown that the cleavage of the C–N bond and the oxidation of thiomorpholine are initiated by reactions involving a cytochrome P450. Journal of Industrial Microbiology & Biotechnology (2001) 26, 2–8. Received 27 December 1999/ Accepted in revised form 08 May 2000