Optimal Conditions For Induction Research Articles

Metabolic engineering of Escherichia coli for efficient degradation of 4-fluorophenol

As a kind of refractory organic pollutant, 4-fluorophenol (4-FP) can be degraded by only a few microorganisms with low efficiency because of the great electron-withdrawing ability of fluorine atoms. So it is necessary to artificially construct engineered strain to improve the degradation efficiency and meet the requirements of pollutant degradation. In this study, four genes (fpdA2, fpdB, fpdC, and fpdD) for 4-FP degradation from Arthrobacter sp. strain IF1 were optimized and synthesized and then reconstructed into Escherichia coli by a multi-monocistronic vector to obtain recombinant BL-fpd that could degrade 4-FP efficiently. Under optimized induction conditions (inducing the strain by 2 g/L L-arabinose and 1 mM IPTG at 37 ℃), BL-fpd could completely degrade 2 mM 4-FP, 4-chlorophenol, 4-bromophenol, and 4-nitrophenol into β-ketoadipate, which could be further metabolized by the bacteria. FpdA2 showed the highest activity towards 4-bromophenol. The strain could completely degrade 1 mM 4-FP in industrial wastewater within 3 h. This study provided a promising strain for the degradation of 4-FP and some other 4-substituted phenols. The construction technologies of multi-monocistronic expression vector may also be used to construct other organic pollutants degrading bacteria.

AOM/DSS Induced Colitis-Associated Colorectal Cancer in 14-Month-Old Female Balb/C and C57/Bl6 Mice-A Pilot Study.

Colitis is a major risk factor for the development of colorectal cancer, leading to colitis-associated colorectal cancer (CAC). The most commonly used animal model to study CAC is the azoxymethane-dextran sulphate-sodium (AOM/DSS) model. The ideal experimental conditions of this model depend on several factors, including the used mouse strain. No data on feasibility and conditions for older mice, e.g., for aging studies, have yet been reported. Thus, we conducted a descriptive, observational pilot study where CAC was induced in 14-month-old female Balb/C and C57/Bl6 mice using 12.5 mg/kg AOM i.p. and three different concentrations of DSS (1, 2, and 3%) in drinking water (ad. lib.). The mice were monitored regularly during the three-month experimental phase. After euthanasia, the colons of the mice were evaluated macroscopically and microscopically. Both the mouse strains showed a DSS-concentration-dependent induction of CAC. Carcinomas were only observed at 3% DSS. The DSS dose was found to be significantly correlated with the histology score and % Ki67 positive cells only in C57/Bl6 mice but not in Balb/C mice, which showed a variable response to the CAC induction. No differences in colon length, weight, or mucin content were observed. Optimal conditions for CAC induction in these aged animals are thus considered to be 3% DSS, as carcinomas did not develop when 2% DSS was used. On the other hand, Balb/C mice reacted severely to 3% DSS, indicating that 2.5% DSS may be the “sweet spot” for future experiments comparing CAC in aged Balb/C and C57/Bl6 mice. This model will allow investigation of the effect of aging on CAC development and therapy.

Induction of meiotic gynogenesis in Lanzhou catfish ( Silurus lanzhouensis ) with heterologous sperm and evidence for female homogamety

As a critical factor in the sustainability of fisheries, gynogenesis can be used to provide a shortcut for the genetic improvement of the endangered and degraded germplasm resources of the important Yellow River fish Silurus lanzhouensis. In this study, ultraviolet-inactivated yellow catfish Pelteobagrus fulvidraco sperm were used to induce meiotic gynogenesis in S. lanzhouensis through cold shock, and the optimal conditions for the genetic inactivation of the sperm and cold shock treatment were selected. Using microsatellite markers and gonadal histology, the genetic purity and sex determination of the gynogenetic offspring of S. lanzhouensis were analysed. The optimal induction conditions were as follows: treatment with P. fulvidraco sperm with 107- µW/cm2 ultraviolet irradiation for 30 min, fertilization of S. lanzhouensis eggs for 6 min and subsequent cold shock treatment at 4–6°C for 25 min. Flow cytometry and microsatellite markers confirmed the successful induction of meiogynogenesis. The homozygosity of the gynogenetic group (0.63) was higher than that of the normal control group (0.32). The sex ratio was determined through gonad histological observations, and 100% of the offspring in the gynogenetic groups were female, strongly indicating that the sex determination system of S. lanzhouensis is the XX/XY-type. In this study, inactivated heterologous sperm were used to successfully obtain meiogynogenetic individuals of S. lanzhouensis, and the study provides an important basis for the establishment of a pure line of these endangered fish and sex-controlled breeding of S. lanzhouensis.

A simple and efficient protocol for hairy root culture of Arabidopsis thaliana

Hairy root culture (HRC) represent a valuable biotechnological tool for the production of plant secondary metabolites. Secondary metabolome study of Arabidopsis thaliana may help to understand the biological roles of various secondary metabolites present in it. The present work deals with the establishment of Agrobacterium rhizogenes strains transformed HRC of A. thaliana with very high transformation frequency resulting in long term hairy root cultures grown in hormone free media. Optimization of culture medium and standardisation of co-cultivation period are the key role players in obtaining high frequency of hairy roots. Four days of preculture in CIM medium and five min of co-cultivation in the bacterial suspension were found to be optimal conditions for root induction. This protocol could become a powerful tool for transcriptomics, metabolomics and proteomics-based studies for different transgenic root lines of A. thaliana.

Molecular cloning, and optimized production and characterization of recombinant cyclodextrin glucanotransferase from Bacillus sp. T1.

The online version contains supplementary material available at 10.1007/s13205-022-03111-8.

Effects of exosomes derived from Trichinella spiralis infective larvae on intestinal epithelial barrier function

Muscle larvae of Trichinella spiralis parasitize the host intestinal epithelium. The mechanisms of exosomes participating in the invasion of T. spiralis muscle larvae are unclear. Hence, the purpose of this study was to explore the effect of exosomes derived from T. spiralis infective larvae (TsExos) on the barrier function of porcine small intestinal epithelial cells (IPEC-J2). First, TsExos were successfully obtained, and their ingestion by epithelial cells was validated. Furthermore, the optimal induction condition was determined by the CCK8 kit, and we found that exposure to 150 μg/mL TsExos for 12/24 h decreased the viability of IPEC-J2 cells by 30%. Based on this outcome, the effects of TsExos on cell biological processes and tight junctions were studied. After coincubation of TsExos and IPEC-J2 cells, the results showed a significant increase in the content of FITC-dextran and in the levels of lactate dehydrogenase (LDH) and reactive oxygen species (ROS). The rate of apoptosis increased by 12.57%, and nuclear pyknosis and nuclear rupture were observed. After the cells were induced by TsExos, the expression of IL-1 was upregulated, but the expression of IL-10, TGF-β, TLR-5, MUC-1 and MUC-2 was downregulated. TsExo induction also led to a decrease in the levels of ZO-1, CLDN-3, and OCLN. In conclusion, TsExos are involved in several cellular biological processes, and they function by disrupting physiological and biochemical processes, hyperactivating innate immunity, and damaging tight junctions.

Surface display of carbonic anhydrase on Escherichia coli for CO2 capture and mineralization

Mineralization catalyzed by carbonic anhydrase (CA) is one of the most promising technologies for capturing CO2. In this work, Escherichia coli BL21(DE3) was used as the host, and the N-terminus of ice nucleation protein (INPN) was used as the carrier protein. Different fusion patterns and vectors were used to construct CA surface display systems for α-carbonic anhydrase (HPCA) from Helicobacter pylori 26695 and α-carbonic anhydrase (SazCA) from Sulfurihydrogenibium azorense. The surface display system in which HPCA was fused with INPN via a flexible linker and intermediate repeat sequences showed higher whole-cell enzyme activity, while the enzyme activity of the SazCA expression system was significantly higher than that of the HPCA expression system. The pET22b vector with the signal peptide PelB was more suitable for the cell surface display of SazCA. Cell fractionation and western-blot analysis indicated that SazCA and INPN were successfully anchored on the cell's outer membrane as a fusion protein. The enzyme activity of the surface display strain E-22b-IRLS (11.43 U·mL−1OD600−1) was significantly higher than that of the intracellular expression strain E-22b-S (8.355 U·mL−1OD600−1) under optimized induction conditions. Compared with free SazCA, E-22b-IRLS had higher thermal and pH stability. The long-term stability of SazCA was also significantly improved by surface display. When the engineered strain and free enzyme were used for CO2 mineralization, the amount of CaCO3 deposition catalyzed by the strain E-22b-IRLS on the surface (241 mg) was similar to that of the free SazCA and was significantly higher than the intracellular expression strain E-22b-S (173 mg). These results demonstrate that the SazCA surface display strain can serve as a whole-cell biocatalyst for CO2 capture and mineralization.

Induction and Proliferation of Callus in the Flower Stalk of Calanthe discolor Lindl. and its Antioxidant Properties

Purpose: This study aims to establish the appropriate conditions for induction and proliferation of callus from the flower stalk of <i>Calanthe discolor</i> Lindl. (CFS) and to analyze the antioxidant properties as well as the skin irritation level of its extracts.Methods: Murashige & Skoog medium was used to induce the callus from CFS with NB0 (α-naphthaleneacetic acid (NAA) 0 mg/L, benzyl adenine (BA) 0 mg/L), NB1 (NAA 0.2 mg/L, BA 0.1 mg/L), NB5 (NAA 0.2 mg/L, BA 0.5 mg/L), and NB10 (0.2 mg/L, BA 1.0 mg/L) as plant growth regulators (PGR). The callus powder was extracted by ultrasonic treatment, and the total phenolic contents (TPC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, and human skin irritation test using callus extracts were performed.Results: As a result of callus induction from CFS with PGR, NB5 showed the highest growth rate of callus with a weight of 4.2±0.3 g. The TPC of the callus extracts NB0, NB1, NB5, and NB10 were 33.71±0.27, 37.59±0.51, 46.26±0.18, and 55.92±0.33 mg GAE/g, respectively. NB10 showed the highest value of DPPH radical scavenging activity than that of NB0, NB1, NB5, and NB10, in various concentrations. CFS callus extract showed very low human skin irritation compared to that of the control group, 10% SLS.Conclusion: The optimal PGR condition for induction of CFS callus was established as NB10. CFS callus extracts can be used as functional materials for cosmetics as it has significant antioxidant activity and low skin irritation value.

Tetraploid induction of Crassostrea hongkongensis and C. sikamea by inhibiting the polar body 1 release in diploid fertilized eggs.

The production of an all-triploid population by mating tetraploid males with diploid females is the best and most fundamental method for the large-scale production of triploid oysters. Obtaining a stable tetraploid population is essential for guaranteed production in industrialized triploid cultivation. C. hongkongensis and C. sikamea are important oyster breeding species in southern China, and have great economic value. However, there are not any published data on inducing tetraploid C. hongkongensis or C. sikamea. Therefore, we investigated tetraploid induction in these two oyster species by inhibiting the PB1 release in diploid fertilized eggs using Cytochalasin B (CB) under 31°C, 15 ‰ salinity. The results confirmed that the optimal tetraploid induction conditions for C. hongkongensis were a CB concentration of 0.50mg/L with induction starting at 9.0min after fertilization, and stopping at 21.0min after fertilization; the induction efficiency index reached 0.123 under these conditions. The optimal tetraploid induction conditions for C. sikamea were a CB concentration of 0.50mg/L, with induction starting at 7.5min after fertilization and stopping at 18min after fertilization; the induction efficiency index could be as high as 0.281 under these conditions. However, we confirmed that the tetraploid rate decreased with larval growth, and no tetraploids were detected in the juvenile period of either C. hongkongensis or C. sikamea. This may be attributed to the very low survival of the tetraploid larvae induced by this method, especially as most tetraploid larvae died during the first three days. In summary, it is simple to directly induce tetraploid C. hongkongensis and C. sikamea larvae by inhibiting the PB1 release of diploid zygotes, but the low survival rate makes it challenging to obtain viable juvenile tetraploids.

VqBGH40a isolated from Chinese wild Vitis quinquangularis degrades trans-piceid and enhances trans-resveratrol

Resveratrol (3,5,4’-trihydroxy-stilbene) is a phytoalexin that can prevent plants from pathogen attacks. Piceid is the glycosylation product of resveratrol and the main storage form of stilbenes in grapevines. Here, we reported the function of a β-glycoside hydrolase gene, VqBGH40a, from the Chinese wild grapevine Vitis quinquangularis accession Danfeng-2 in the regulation of plant resistance to powdery mildew (Uncinula necator). VqBGH40a belonging to β-glycoside hydrolase family 1 encoded 506 amino acids and was located on the cytomembrane. Its optimal induction condition was 28 or 30℃, for 4 h, with 0.1 mM IPTG in a prokaryotic expression system. Enzyme activity detection showed that purified VqBGH40a could hydrolyze trans-piceid to form trans-resveratrol in vitro. VqBGH40a was transiently overexpressed in Danfeng-2 leaves and then artificially inoculated with powdery mildew showed that VqBGH40a protein could hydrolyze trans-piceid in vivo. Additionally, a comparative family analysis between VqBGH40a and 38 VviBGHs was performed. Overall, these results demonstrate that VqBGH40a can hydrolyze trans-piceid, enhance trans-resveratrol content, and participate in the defense mechanism of grapevine against powdery mildew.

Induction of Polyploidy and Metabolic Profiling in the Medicinal Herb Wedelia chinensis.

Wedelia chinensis, which belongs to the Asteraceae family, is a procumbent, perennial herb. It has medicinal anti-inflammatory properties and has been traditionally used as folk medicine in East and South Asia for treating fever, cough and phlegm. In Taiwan, W. chinensis is a common ingredient of herbal tea. Previous studies showed that the plant leaves contain four major bioactive compounds, wedelolactone, demethylwedelolactone, luteolin and apigenin, that have potent antihepatoxic activity, and are thus used as major ingredients in phytopharmaceutical formulations. In this study, we set up optimal conditions for induction of ploidy in W. chinensis. Ploidy can be an effective method of increasing plant biomass and improving medicinal and ornamental characteristics. By using flow cytometry and chicken erythrocyte nuclei as a reference, the DNA content (2C) or genome size of W. chinensis was determined to be 4.80 picograms (pg) in this study for the first time. Subsequently, we developed the successful induction of five triploid and three tetraploid plants by using shoot explants treated with different concentrations (0, 0.25, 0.5, 1, 1.5, 2 g/L) of colchicine. No apparent morphological changes were observed between these polyploid plants and the diploid wild-type (WT) plant, except that larger stomata in leaves were found in all polyploid plants as compared to diploid WT. Ultra-performance liquid chromatography coupled with tandem mass spectrometry was used to quantify the four index compounds (wedelolactone, demethylwedelolactone, luteolin, apigenin) in these polyploid plants, and fluctuating patterns were detected. This is the first report regarding polyploidy in the herbal plant W. chinensis.

Induction of spontaneous phenotype conversion in Ralstonia solanacearum by addition of iron compounds in liquid medium

Ralstonia solanacearum is a soil-borne pathogen that causes bacterial wilt in plants. The wild-type strain of R. solanacearum undergoes spontaneous phenotype conversion (PC), from a fluidal to non-fluidal colony morphology. PC mutants are non-pathogenic due to reduced virulence factors, and can control wilt diseases as biological control agents. The induction factors of PC in R. solanacearum are currently unclear. Here, we investigated the effect of iron treatment on bacterial growth of wild-type strain and PC mutant, and PC of the wild-type strain in liquid medium. Interestingly, PC was frequently induced in the single cultured wild-type strain by iron treatment; however, PC was not induced in the co-culture. In a co-culture of both strains, the PC mutant showed increased growth compared to the wild-type strain by iron treatment. Furthermore, we investigated the effects of iron treatment on the bacterial growth and PC of the wild-type strain under different culture conditions of medium type (MM broth, BG broth, and water medium), iron compounds, and pH. In BG broth, PC occurred frequently regardless of iron treatment. In MM broth, the optimal conditions for high frequency induction of PC by iron treatments were treatment of iron (III) EDTA, and under pH 7–8. Conversely, PC was not induced by iron treatment in water medium and in MM broth under pH 5 conditions. Common to the culture conditions wherein PC was not induced by iron treatment, the bacterial density of the wild-type strain was as low as 106 CFU mL−1 or less. Finally, we investigated the effects on bacterial growth and PC of the wild-type strain by the iron treatment and addition of culture filtrate after cultivation of the wild-type strain at high concentration. In medium containing only the culture filtrate, PC did not occur. However, in medium containing the culture filtrate and iron, PC occurred frequently. Our results thus suggest that high-density growth of the wild-type strain as well as the presence of iron are involved in inducing PC in R. solanacearum.

Expression, purification, and characterization of pneumococcal PsaA-PspA fusion protein

Streptococcus pneumoniae is a gram-positive bacterial pathogen causing invasive pneumonia, meningitis, otitis media, and bacteremia. Owing to the current pitfalls of polysaccharide and polysaccharide-conjugate vaccines, protein vaccines are considered promising candidates against pneumonia. Pneumococcal surface protein A (PspA) and pneumococcal surface adhesin A (PsaA) are virulence proteins showing good immunogenicity and protective effects against S. pneumoniae strains in mice. In this study, we expressed the fusion protein PsaA-PspA, which consists of PsaA and the N-terminal region of PspA family 1 and 2, in Escherichia coli. We describe a novel and effective method to purify PsaA-PspA using hydroxyapatite and two-step chromatography. After determining the optimal induction conditions and a series of purification steps, we obtained PsaA-PspA fusion protein with over 95% purity at a final yield of 22.44% from the starting cell lysate. The molecular weight of PsaA-PspA was approximately 83.6 kDa and its secondary structure was evaluated by circular dichroism. Immunization with the purified protein induced high levels of IgG antibodies in mice. Collectively, these results demonstrate that our purification method can effectively produce high-purity PsaA-PspA fusion protein with biological activity and chemical integrity, which can be widely applied to the purification of other PspA subclass proteins.

Generation and optimization of highly pure motor neurons from human induced pluripotent stem cells via lentiviral delivery of transcription factors.

Generation of neurons from human induced pluripotent stem cells (hiPSCs) overcomes the limited access to human brain samples and greatly facilitates the progress of research in neurological diseases. However, it is still a challenge to generate a particular neuronal subtype with high purity and yield for determining the pathogenesis of diseased neurons using biochemical approaches. Motor neurons (MNs) are a specialized neuronal subtype responsible for governing both autonomic and volitional movement. Dysfunctions in MNs are implicated in a variety of movement diseases, such as amyotrophic lateral sclerosis (ALS). In this study, we generated functional MNs from human iPSCs via lentiviral delivery of transcription factors. Moreover, we optimized induction conditions by using different combinations of transcription factors and found that a single lentiviral vector expressing three factors [neurogenin-2 (NGN2), insulin gene enhancer 1 (ISL1), and LIM/homeobox 3 (LHX3)] is necessary and sufficient to induce iPSC-derived MNs (iPSC-MNs). These MNs robustly expressed general neuron markers [microtubule-associated protein 2 (MAP2), neurofilament protein (SMI-32), and tubulin β-3 class III (TUBB3)] and MN-specific markers [HB9 and choline acetyltransferase (ChAT)] and showed electrical maturation and firing of action potentials within 3 wk. This approach significantly improved the neuronal survival, yield, and purity, making it feasible to obtain abundant materials for biochemical studies in modeling movement diseases.

Highly secretory expression of recombinant cowpea chlorotic mottle virus capsid proteins in Pichia pastoris and in-vitro encapsulation of ruthenium nanoparticles for catalysis

The applications of viral protein cages have expanded rapidly into the fields of bionanotechnology and materials science. However, the low-cost production of viral capsid proteins (CPs) on a large scale is always a challenge. Herein, we develop a highly efficient expression system by constructing recombinant Pichia pastoris cells as a “factory” for the secretion of soluble cowpea chlorotic mottle virus (CCMV) CPs. Under optimal induction conditions (0.9 mg/mL of methanol concentration at 30 °C for 96 h), a high yield of approximately 95 mg/L of CCMV CPs was harvested from the fermentation supernatant with CPs purity >90%, which has significantly simplified the rest of the purification process. The resultant CPs are employed to encapsulate Ruthenium (Ru) nanoparticles (NPs) via in-vitro self-assembly to prepare hybrid nanocatalyst, i.e. Ru@virus-like particles (VLPs). The catalytic activity over Ru@VLPs was evaluated by reducing 4-nitrophenol (4-NP) to 4-aminophenol (4-AP). The results indicate that, with the protection of protein cages, Ru NPs were highly stabilized during the catalytic reaction. This results in enhanced catalytic activity (reaction rate constant k = 0.14 min−1) in comparison with unsupported citrate-stabilized Ru NPs (Ru-CA) (k = 0.08 min−1). Additionally, comparatively lower activation energy over Ru@VLPs (approximately 32 kJ/mol) than that over Ru-CA (approximately 39 kJ/mol) could be attributed to the synergistic effect between Ru NPs and some functional groups such as amino groups (–NH2) on CPs that weakened the activation barrier of 4-NP reduction. Therefore, enhanced activity and decreased activation energy over Ru@VLPs demonstrated the superiority of Ru@VLPs to unsupported Ru-CA.

Application of the cbh1 promoter in Mortierella alpina and optimization of induction conditions.

Mortierella alpina has gained remarkable interest due to its high capacity for arachidonic acid (AA) production and potential for eicosapentaenoic acid (EPA) production recently. However, the development of genetically modified strains is limited by lacking inducible promoters, which can express genes conditionally. Here the inducible promoter of cellobiohydrolase (Pcbh1) was utilized in M. alpina and the gene oPpFADS17 encoding ω-3 fatty acid desaturase was selected as the reporter gene. Under conditions with inducer, expression of this gene enables M. alpina to produce EPA at room temperature, while no EPA was detected without inducer. We then optimized the induction conditions. The results demonstrated that the optimal induction condition was broth medium with 1% avicel as the inducer and 5% glucose as extra carbon source and the transcription level of the reporter gene was increasing with the extension of induction time. Successful application of Pcbh1 in M. alpina would significantly contribute to the steerable system to construct engineered strains for industrial production of microbial oils. SIGNIFICANCE AND IMPACT OF THE STUDY: Mortierella alpina is a commercial strain for production of polyunsaturated fatty acids. Genetic engineering strategies based on M. alpina require the development of inducible promoters to regulate gene expression conditionally at specific times. However, available inducible promoters for M. alpina were limited. In this study, we explore the feasibility of inducible cbh1 promoter in M. alpina and determined the optimal induction condition, which accelerates the genetic manipulation of M. alpina. Besides, high transcriptional levels of the reporter gene under the control of Pcbh1 showed that Pcbh1 is a strong inducible promoter for M. alpina.

In vitro polyploid induction using colchicine for Zingiber Officinale Roscoe cv. ‘Fengtou’ ginger

‘Fengtou’ ginger is a high quality ginger (Zingiber Officinale Roscoe) cultivar only planted in Laifeng County, Enshi, Huhei, China, and is a National Product of Geographical Indication originated. ‘Fengtou’ ginger yield is low compared with most ginger. Therefore, it is imperative to apply breeding methods to improve yield. One strategy to accelerate breeding is polyploid induction, which could improve yield and maintain quality. In this study, different concentrations of colchicine (50, 100, 150, 200 mg/L) and different durations of exposure (3, 5, 7 days) were applied to stem segments of adventitious buds in ginger. Among obtained polyploidy plantlets were identified by flow cytometric analysis. The optimal induction condition of tetraploids was that the concentrations of colchicine was 150 mg/L, induced for 7 days, the chromosome doubling rate reached 18%. The tetraploid plants achieved were larger than their diploid plants for leaf length, leaf width, leaf thickness, stem diameter and guard cell. The soluble sugar, soluble protein, proline and other substances were also significant difference, especially carotenoids concentration was 1.375 times in contrast with diploid plants, which showed that tetraploids ginger had potentiality to increase yield and better adaptability. We explored the polyploid induction system of Zingiber Officinale Roscoe cv. ‘Fengtou’ ginger. Studies of morphological characteristics and secondary metabolites have shown that tetraploids ginger have potentiality to increase yield and better adaptability.

Plant regeneration in Vernicia fordii from apical bud

Vernicia fordii is a woody plant species with important economic value, its seeds could be used to produce biodiesel and the oil extracted from them serve many kinds of industrial applications. In this study, we established a regeneration system of V. fordii for the first time using apical buds as explants on the Murashige and Skoog (MS) medium supplemented with the plant growth regulators, 6-benzylaminopurine (6-BA) and 3-indolebutyric acid (IBA). The optimal condition for adventitious bud induction was MS medium containing 1.5 mg/L 6-BA and 0.01 mg/L IBA, which produced a satisfactory induction efficiency. In subculture, the MS medium containing 1.0 mg/L 6-BA and 0.01 mg/L IBA was the optimum selection when considering both proliferation rate and tissue growth status. The adventitious shoots inoculated on 1/2 MS medium containing 0.5 mg/L IBA could produce strong roots and 96.4% of the regenerated plants survived after transplanting.

Construction of a novel sugar alcohol-inducible expression system in Bacillus licheniformis.

Bacillus licheniformis is an important industrial microorganism that can utilize a wide range of biomass. However, the lack of expression elements in B. licheniformis, especially regulated promoters, significantly restricts its applications. In this study, two promoters involved in the sugar alcohol uptake pathway, PmtlA and PmtlR, were characterized and developed as regulated promoters for expression. The results showed that mannitol, mannose, sorbitol, sorbose, and arabinose can act as inducers to activate expression from PmtlA at different levels. The induction by sorbitol was the strongest, and the optimal induction conditions were 15g/L sorbitol during mid-logarithmic growth at 28°C. In this work, the palindrome-like sequence 'TTGTCA-cacggctcc-TGCCAA' in PmtlA was identified as the binding site of the MtlR protein. This study helps to enrich the known inducible expression systems in B. licheniformis.

Harnessing β-estradiol inducible expression system to overproduce nervonic acid in Saccharomyces cerevisiae

As a very long chain monounsaturated fatty acid, nervonic acid (NA) holds great application potential for brain diseases. 3-ketoacyl-CoA synthase (KCS) with substrate specificity plays a key role in the carbon chain elongation cycle for NA biosynthesis. A tunable and sensitive single-gene expression regulation is necessary to overproduce NA in yeast cell. Herein, β-estradiol inducible expression system (EIES) was employed to augment NA biosynthesis in Saccharomyces cerevisiae. EIES consisted of two recombinant vectors: pRS416-PGal1-GFP-TCYC1 and YEplac112-PTEF1-ZEV-TCYC1. The following was the optimized induction conditions of EIES in SD medium: 0.1 μM β-estradiol, 15 min of heat induction at 33 ℃ every 8 h within first 32 h. Using EIES to enhance KCS and ELOVL1 expression, NA contents in the recombinants increased by 332 % and 101 % respectively, compared with the control strain using strong PGK promoter. NA levels were further improved by knockout of elo2 in the recombinants expressing KCS or ELOVL1. This study suggested that EIES could be a promising tool for enhancing the biosynthesis of desired metabolites.