RESUMEN
Coproporphyrin III (CP III), a natural porphyrin derivative, has extensive applications in the biomedical and material industries. S. cerevisiae has previously been engineered to highly accumulate the CP III precursor 5-aminolevulinic acid (ALA) through the C4 pathway. In this study, a combination of cytoplasmic metabolic engineering and mitochondrial compartmentalization was used to enhance CP III production in S. cerevisiae. By integrating pathway genes into the chromosome, the CP III titer gradually increased to 32.5 ± 0.5 mg/L in shake flask cultivation. Nevertheless, increasing the copy number of pathway genes did not consistently enhance CP III synthesis. Hence, the partial synthesis pathway was compartmentalized in mitochondria to evaluate its effectiveness in increasing CP III production. Subsequently, by superimposing the mitochondrial compartmentalization strategy on cytoplasmic metabolic engineered strains, the CP III titer was increased to 64.3 ± 1.9 mg/L. Furthermore, augmenting antioxidant pathway genes to reduce reactive oxygen species (ROS) levels effectively improved the growth of engineered strains, resulting in a further increase in the CP III titer to 82.9 ± 1.4 mg/L. Fed-batch fermentations in a 5 L bioreactor achieved a titer of 402.8 ± 9.3 mg/L for CP III. This study provides a new perspective on engineered yeast for the microbial production of porphyrins.
RESUMEN
Rehmannia glutinosa is one of the commonly used Chinese herbal medicines, which has activities of heat-clearing,blood-cooling, Yin-nourishing, and body fluid-promoting. Iridoid glycosides are the main bioactive in R. glutinosa. Iridoid oxidase is a key rate-limiting enzyme in the biosynthetic pathway of iridoid glycosides. In this study, an iridoid oxidase gene Rg IO was screened based on the transcriptome data, followed by bioinformatics analysis, expression characteristic detection, and subcellular localization analysis. The results show that the coding region of Rg IO is 1 536 bp, with 511 amino acids encoded, and the molecular weight is about 58 258. 01. The protein sequence of Rg IO contains the conserved domains and motifs of cytochrome P450 oxidases. Rg IO has the highest sequence identities with its ortholog proteins in Striga asiatica, Striga hermonthica, and Centranthera grandiflora and has good sequence identities(77. 28%) with Catharanthus roseus Cr IO. Rg IO shows specific expression in the leaf of R. glutinosa. In response to MeJA induction, the expression of MeJA in leaves and roots after treatment increases by 3. 15 and 1. 3 times at 3 h and 6 h,respectively. The result of subcellular localization shows that Rg IO is distributed in the endoplasmic reticulum. Agrobacterium-mediated transient expression of Rg IO gene in leaves of R. glutinosa makes the content of catalpol increase by 0. 82 times compared with the transient expression of the empty vector. This study provides a key target gene for the molecular regulation and biosynthesis of catalpol in R. glutinosa and lays a foundation for revealing the complete biosynthetic pathway of catalpol.
Asunto(s)
Clonación Molecular , Proteínas de Plantas , Rehmannia , Rehmannia/genética , Rehmannia/enzimología , Rehmannia/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/química , Regulación de la Expresión Génica de las Plantas , Filogenia , Secuencia de AminoácidosRESUMEN
Heme has attracted considerable attention due to its indispensable biological roles and applications in healthcare and artificial foods. The development and utilization of edible microorganisms instead of animals to produce heme is the most promising method to promote the large-scale industrial production and safe application of heme. However, the cytotoxicity of heme severely restricts its efficient synthesis by microorganisms, and the cytotoxic mechanism is not fully understood. In this study, the effect of heme toxicity on Saccharomyces cerevisiae was evaluated by enhancing its synthesis using metabolic engineering. The results showed that the accumulation of heme after the disruption of heme homeostasis caused serious impairments in cell growth and metabolism, as demonstrated by significantly poor growth, mitochondrial damage, cell deformations, and chapped cell surfaces, and these features which were further associated with substantially elevated reactive oxygen species (ROS) levels within the cell (mainly H2O2 and superoxide anion radicals). To improve cellular tolerance to heme, 5 rounds of laboratory evolution were performed, increasing heme production by 7.3-fold and 4.2-fold in terms of the titer (38.9 mg/L) and specific production capacity (1.4 mg/L/OD600), respectively. Based on comparative transcriptomic analyses, 32 genes were identified as candidates that can be modified to enhance heme production by more than 20% in S. cerevisiae. The combined overexpression of 5 genes (SPS22, REE1, PHO84, HEM4 and CLB2) was shown to be an optimal method to enhance heme production. Therefore, a strain with enhanced heme tolerance and ROS quenching ability (R5-M) was developed that could generate 380.5 mg/L heme with a productivity of 4.2 mg/L/h in fed-batch fermentation, with S. cerevisiae strains being the highest producers reported to date. These findings highlight the importance of improving heme tolerance for the microbial production of heme and provide a solution for efficient heme production by engineered yeasts.
RESUMEN
BACKGROUND: Social needs inhibit receipt of timely medical care. Social needs screening is a vital part of comprehensive cancer care, and patient navigators are well-positioned to screen for and address social needs. This mixed methods project describes social needs screening implementation in a prospective pragmatic patient navigation intervention trial for minoritized women newly diagnosed with breast cancer. METHODS: Translating Research Into Practice (TRIP) was conducted at five cancer care sites in Boston, MA from 2018 to 2022. The patient navigation intervention protocol included completion of a social needs screening survey covering 9 domains (e.g., food, transportation) within 90 days of intake. We estimated the proportion of patients who received a social needs screening within 90 days of navigation intake. A multivariable log binomial regression model estimated the adjusted rate ratios (aRR) and 95% confidence intervals (CI) of patient socio-demographic characteristics and screening delivery. Key informant interviews with navigators (n = 8) and patients (n = 21) assessed screening acceptability and factors that facilitate and impede implementation. Using a convergent, parallel mixed methods approach, findings from each data source were integrated to interpret study results. RESULTS: Patients' (n = 588) mean age was 59 (SD = 13); 45% were non-Hispanic Black and 27% were Hispanic. Sixty-nine percent of patients in the navigators' caseloads received social needs screening. Patients of non-Hispanic Black race/ethnicity (aRR = 1.25; 95% CI = 1.06-1.48) and those with Medicare insurance (aRR = 1.13; 95% CI = 1.04-1.23) were more likely to be screened. Screening was universally acceptable to navigators and generally acceptable to patients. Systems-based supports for improving implementation were identified. CONCLUSIONS: Social needs screening was acceptable, yet with modest implementation. Continued systems-based efforts to integrate social needs screening in medical care are needed.
Asunto(s)
Neoplasias de la Mama , Navegación de Pacientes , Humanos , Femenino , Neoplasias de la Mama/diagnóstico , Persona de Mediana Edad , Estudios Prospectivos , Anciano , Evaluación de Necesidades , Boston , AdultoRESUMEN
BACKGROUND: Marine microalgae (phytoplankton) mediate almost half of the worldwide photosynthetic carbon dioxide fixation and therefore play a pivotal role in global carbon cycling, most prominently during massive phytoplankton blooms. Phytoplankton biomass consists of considerable proportions of polysaccharides, substantial parts of which are rapidly remineralized by heterotrophic bacteria. We analyzed the diversity, activity, and functional potential of such polysaccharide-degrading bacteria in different size fractions during a diverse spring phytoplankton bloom at Helgoland Roads (southern North Sea) at high temporal resolution using microscopic, physicochemical, biodiversity, metagenome, and metaproteome analyses. RESULTS: Prominent active 0.2-3 µm free-living clades comprised Aurantivirga, "Formosa", Cd. Prosiliicoccus, NS4, NS5, Amylibacter, Planktomarina, SAR11 Ia, SAR92, and SAR86, whereas BD1-7, Stappiaceae, Nitrincolaceae, Methylophagaceae, Sulfitobacter, NS9, Polaribacter, Lentimonas, CL500-3, Algibacter, and Glaciecola dominated 3-10 µm and > 10 µm particles. Particle-attached bacteria were more diverse and exhibited more dynamic adaptive shifts over time in terms of taxonomic composition and repertoires of encoded polysaccharide-targeting enzymes. In total, 305 species-level metagenome-assembled genomes were obtained, including 152 particle-attached bacteria, 100 of which were novel for the sampling site with 76 representing new species. Compared to free-living bacteria, they featured on average larger metagenome-assembled genomes with higher proportions of polysaccharide utilization loci. The latter were predicted to target a broader spectrum of polysaccharide substrates, ranging from readily soluble, simple structured storage polysaccharides (e.g., laminarin, α-glucans) to less soluble, complex structural, or secreted polysaccharides (e.g., xylans, cellulose, pectins). In particular, the potential to target poorly soluble or complex polysaccharides was more widespread among abundant and active particle-attached bacteria. CONCLUSIONS: Particle-attached bacteria represented only 1% of all bloom-associated bacteria, yet our data suggest that many abundant active clades played a pivotal gatekeeping role in the solubilization and subsequent degradation of numerous important classes of algal glycans. The high diversity of polysaccharide niches among the most active particle-attached clades therefore is a determining factor for the proportion of algal polysaccharides that can be rapidly remineralized during generally short-lived phytoplankton bloom events. Video Abstract.
Asunto(s)
Flavobacteriaceae , Microalgas , Fitoplancton/genética , Fitoplancton/metabolismo , Eutrofización , Polisacáridos/metabolismo , Flavobacteriaceae/metabolismo , Microalgas/metabolismoRESUMEN
Ergothioneine (EGT) is a rare thiohistidine derivative with exceptional antioxidant properties. The blood level of EGT is considered highly reliable predictors for cardiovascular diseases and mortality, yet animals lack the ability to synthesize this compound. Free plasmids have been previously used to overexpress genes involved in the EGT biosynthetic pathway of Mycolicibacterium neoaurum. Here, we tentatively introduced a putative transporter gene mfsT1 into high-copy plasmids and sharply increased the ratio of extracellular EGT concentration from 18.7% to 44.9%. Subsequently, an additional copy of egtABCDE, hisG, and mfsT1 was inserted into the genome with a site-specific genomic integration tool of M. neoaurum, leading a 2.7 times increase in EGT production. Co-enhancing the S-adenosyl-L-methionine regeneration pathway, or alternatively, the integration of three copies of egtABCDE, hisG and mfsT1 into the genome further increased the total EGT yield by 16.1% (64.6 mg/L) and 21.7% (67.7 mg/L), respectively. After 168-h cultivation, the highest titer reached 85.9 mg/L in the latter strain with three inserted copies. This study provided a solid foundation for genome engineering to increase the production of EGT in M. neoaurum.
Asunto(s)
Ergotioneína , Mycobacteriaceae , Animales , Ergotioneína/genética , Ergotioneína/metabolismo , Antioxidantes/metabolismoRESUMEN
Sepsis, a life-threatening health issue, lacks effective medicine targeting the septic response. In China, treatment combining the intravenous herbal medicine XueBiJing with conventional procedures reduces the 28-day mortality of critically ill patients by modulating septic response. In this study, we identified the combined active constituents that are responsible for the XueBiJing's anti-sepsis action. Sepsis was induced in rats by cecal ligation and puncture (CLP). The compounds were identified based on their systemic exposure levels and anti-sepsis activities in CLP rats that were given an intravenous bolus dose of XueBiJing. Furthermore, the identified compounds in combination were assessed, by comparing with XueBiJing, for levels of primary therapeutic outcome, pharmacokinetic equivalence, and pharmacokinetic compatibility. We showed that a total of 12 XueBiJing compounds, unchanged or metabolized, circulated with significant systemic exposure in CLP rats that received XueBiJing. Among these compounds, hydroxysafflor yellow A, paeoniflorin, oxypaeoniflorin, albiflorin, senkyunolide I, and tanshinol displayed significant anti-sepsis activities, which involved regulating immune responses, inhibiting excessive inflammation, modulating hemostasis, and improving organ function. A combination of the six compounds, with the same respective doses as in XueBiJing, displayed percentage survival and systemic exposure in CLP rats similar to those by XueBiJing. Both the combination and XueBiJing showed high degrees of pharmacokinetic compatibility regarding interactions among the six active compounds and influences of other circulating XueBiJing compounds. The identification of XueBiJing's pharmacologically significant constituents supports the medicine's anti-sepsis use and provides insights into a polypharmacology-based approach to develop medicines for effective sepsis management.
Asunto(s)
Medicamentos Herbarios Chinos , Ratas Sprague-Dawley , Sepsis , Animales , Sepsis/tratamiento farmacológico , Medicamentos Herbarios Chinos/farmacología , Medicamentos Herbarios Chinos/administración & dosificación , Medicamentos Herbarios Chinos/uso terapéutico , Medicamentos Herbarios Chinos/farmacocinética , Masculino , Ratas , Administración IntravenosaRESUMEN
Indigoidine, as a kind of natural blue pigment, is widely used in textiles, food, and pharmaceuticals and is mainly synthesized from l-glutamine via a condensation reaction by indigoidine synthetases, most of which originates from Streptomyces species. However, due to the complex metabolic switches of Streptomyces, most of the researchers choose to overexpress indigoidine synthetases in the heterologous host to achieve high-level production of indigoidine. Considering the advantages of low-cost culture medium and simple culture conditions during the large-scale culture of Streptomyces, here, an updated regulation system derived from the Streptomyces self-sustaining system, constructed in our previous study, was established for the highly efficient production of indigoidine in Streptomyces lividans TK24. The updated system was constructed via promoter mining and σhrdB expression optimization, and this system was applied to precisely and continuously regulate the expression of indigoidine synthetase IndC derived from Streptomyces albus J1704. Finally, the engineered strain was cultured with cheap industrial glycerol as a supplementary carbon source, and 14.3 and 46.27 g/L indigoidine could be achieved in a flask and a 4 L fermentor, respectively, reaching the highest level of microbial synthesis of indigoidine. This study will lay a foundation for the industrial application of Streptomyces cell factories to produce indigoidine.
Asunto(s)
Piperidonas , Streptomyces , Streptomyces/genética , Streptomyces/metabolismo , Piperidonas/metabolismo , Regiones Promotoras Genéticas , Péptido Sintasas/genéticaRESUMEN
BACKGROUND: Harnessing engineered Mycolicibacteria to convert cheap phytosterols into valuable steroid synthons is a basic way in the industry for the production of steroid hormones. Thus, C-19 and C-22 steroids are the two main types of commercial synthons and the products of C17 side chain degradation of phytosterols. During the conversion process of sterols, C-19 and C-22 steroids are often produced together, although one may be the main product and the other a minor byproduct. This is a major drawback of the engineered Mycolicibacteria for industrial application, which could be attributed to the co-existence of androstene-4-ene-3,17-dione (AD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (HBC) sub-pathways in the degradation of the sterol C17 side chain. Since the key mechanism underlying the HBC sub-pathway has not yet been clarified, the above shortcoming has not been resolved so far. RESULTS: The key gene involved in the putative HBC sub-pathway was excavated from the genome of M. neoaurum by comparative genomic analysis. Interestingly, an aldolase- encoding gene, atf1, was identified to be responsible for the first reaction of the HBC sub-pathway, and it exists as a conserved operon along with a DUF35-type gene chsH4, a reductase gene chsE6, and a transcriptional regulation gene kstR3 in the genome. Subsequently, atf1 and chsH4 were identified as the key genes involved in the HBC sub-pathway. Therefore, an updated strategy was proposed to develop engineered C-19 or C-22 steroid-producing strains by simultaneously modifying the AD and HBC sub-pathways. Taking the development of 4-HBC and 9-OHAD-producing strains as examples, the improved 4-HBC-producing strain achieved a 20.7 g/L production titer with a 92.5% molar yield and a 56.4% reduction in byproducts, and the improved 9-OHAD producing strain achieved a 19.87 g/L production titer with a 94.6% molar yield and a 43.7% reduction in byproduct production. CONCLUSIONS: The excellent performances of these strains demonstrated that the primary operon involved in the HBC sub-pathway improves the industrial strains in the conversion of phytosterols to steroid synthons.
RESUMEN
Many marine bacteria are difficult to culture because they are dormant, rare or found in low-abundances. Enrichment culturing has been widely tested as an important strategy to isolate rare or dormant microbes. However, many more mechanisms remain uncertain. Here, based on 16S rRNA gene high-throughput sequencing and metabolomics technology, it was found that the short-chain fatty acids (SCFAs) in metabolites were significantly correlated with uncultured bacterial groups during enrichment cultures. A pure culture analysis showed that the addition of SCFAs to media also resulted in high efficiency for the isolation of uncultured strains from marine sediments. As a result, 238 strains belonging to 10 phyla, 26 families and 82 species were successfully isolated. Some uncultured rare taxa within Chlorobi and Kiritimatiellaeota were successfully cultured. Amongst the newly isolated uncultured microbes, most genomes, e.g. bacteria, possess SCFA oxidative degradation genes, and these features might aid these microbes in better adapting to the culture media. A further resuscitation analysis of a viable but non-culturable (VBNC) Marinilabiliales strain verified that the addition of SCFAs could break the dormancy of Marinilabiliales in 5 days, and the growth curve test showed that the SCFAs could shorten the lag phase and increase the growth rate. Overall, this study provides new insights into SCFAs, which were first studied as resuscitation factors in uncultured marine bacteria. Thus, this study can help improve the utilisation and excavation of marine microbial resources, especially for the most-wanted or key players. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00187-w.
RESUMEN
The engineered probiotic Escherichia coli Nissle 1917 (EcN) is expected to be employed in the diagnosis and treatment of various diseases. However, the introduced plasmids typically require antibiotics to maintain genetic stability, and the cryptic plasmids in EcN are usually eliminated to avoid plasmid incompatibility which may change the inherent probiotic characteristics. Here, we provided a simple design to minimize the genetic change of probiotics by eliminating native plasmids and reintroducing the recombinants carrying functional genes. Specific insertion sites in the vectors showed significant differences in the expression of fluorescence proteins. Selected integration sites were applied in the de novo synthesis of salicylic acid, leading to a titer of 142.0 ± 6.0 mg/L in a shake flask with good production stability. Additionally, the design successfully realized the biosynthesis of ergothioneine (45 mg/L) by one-step construction. This work expands the application scope of native cryptic plasmids to the easy construction of functional pathways. KEY POINTS: ⢠Cryptic plasmids of EcN were designed to express exogenous genes ⢠Insertion sites with different expression intensities in cryptic plasmids were provided ⢠Target products were stably produced by engineering cryptic plasmids.
Asunto(s)
Antibacterianos , Probióticos , Antibacterianos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Plásmidos/genéticaRESUMEN
Endocrine-disrupting compounds (EDCs) are widely distributed in the environment. Here, we present a CRISPR/Cas12a (CAS) biosensor based on DNA aptamers for point-of-care detection of EDCs. Two typical EDCs, 17ß-estradiol (E2) and bisphenol A (BPA), were selected to be detected by the CAS biosensors via the plug-and-play of their DNA aptamers. The results indicated that the performance of the CAS biosensors can be well regulated by controlling the trans-cleavage activity of Cas12a on a single-stranded DNA reporter and optimizing the sequence and ratio of DNA aptamer and activator DNA. Ultimately, two reliable and specific biosensors were developed, with the linear range and limit of detection of 0.2-25 nM and 0.08 nM for E2 and of 0.1-250 nM and 0.06 nM for BPA, respectively. Compared to the existing detection methods, the CAS biosensors showed higher reliability and sensitivity with simple operation, short detection time, and no costly equipment.
Asunto(s)
Aptámeros de Nucleótidos , Técnicas Biosensibles , Aptámeros de Nucleótidos/genética , Sistemas CRISPR-Cas , Reproducibilidad de los Resultados , Estradiol , Técnicas Biosensibles/métodosRESUMEN
BACKGROUND: The conversion of phytosterols to steroid synthons by engineered Mycolicibacteria comprises one of the core steps in the commercial production of steroid hormones. This is a complex oxidative catabolic process, and taking the production of androstenones as example, it requires about 10 equivalent flavin adenine dinucleotide (FAD). As the high demand for FAD, the insufficient supply of FAD may be a common issue limiting the conversion process. RESULTS: We substantiated, using the production of 9α-hydroxy-4-androstene-3,17-dione (9-OHAD) as a model, that increasing intracellular FAD supply could effectively increase the conversion of phytosterols into 9-OHAD. Overexpressing ribB and ribC, two key genes involving in FAD synthesis, could significantly enhance the amount of intracellular FAD by 167.4% and the production of 9-OHAD by 25.6%. Subsequently, styrene monooxygenase NfStyA2B from Nocardia farcinica was employed to promote the cyclic regeneration of FAD by coupling the oxidation of nicotinamide adenine dinucleotide (NADH) to NAD+, and the production of 9-OHAD was further enhanced by 9.4%. However, the viable cell numbers decreased by 20.1%, which was attributed to sharply increased levels of H2O2 because of the regeneration of FAD from FADH2. Thus, we tried to resolve the conflict between FAD regeneration and cell growth by the overexpression of catalase and promotor replacement. Finally, a robust strain NF-P2 was obtained, which could produce 9.02 g/L 9-OHAD after adding 15 g/L phytosterols with productivity of 0.075 g/(L h), which was 66.7% higher than that produced by the original strain. CONCLUSIONS: This study highlighted that the cofactor engineering, including the supply and recycling of FAD and NAD+ in Mycolicibacterium, should be adopted as a parallel strategy with pathway engineering to improve the productivity of the industrial strains in the conversion of phytosterols into steroid synthons.
RESUMEN
BACKGROUND: Macroalgal epiphytic microbial communities constitute a rich resource for novel enzymes and compounds, but studies so far largely focused on tag-based microbial diversity analyses or limited metagenome sequencing of single macroalgal species. RESULTS: We sampled epiphytic bacteria from specimens of Ulva sp. (green algae), Saccharina sp. (brown algae), Grateloupia sp. and Gelidium sp. (both red algae) together with seawater and sediment controls from a coastal reef in Weihai, China, during all seasons. Using 16S rRNA amplicon sequencing, we identified 14 core genera (consistently present on all macroalgae), and 14 dominant genera (consistently present on three of the macroalgae). Core genera represented ~ 0.7% of all genera, yet accounted for on average 51.1% of the bacterial abundances. Plate cultivation from all samples yielded 5,527 strains (macroalgae: 4,426) representing 1,235 species (685 potentially novel). Sequencing of selected strains yielded 820 non-redundant draft genomes (506 potentially novel), and sequencing of 23 sampled metagenomes yielded 1,619 metagenome-assembled genomes (MAGs), representing further 1,183 non-redundant genomes. 230 isolates and 153 genomes were obtained from the 28 core/dominant genera. We analyzed the genomic potential of phycosphere bacteria to degrade algal polysaccharides and to produce bioactive secondary metabolites. We predicted 4,451 polysaccharide utilization loci (PULs) and 8,810 biosynthetic gene clusters (BGCs). These were particularly prevalent in core/dominant genera. CONCLUSIONS: Our metabolic annotations and analyses of MAGs and genomes provide new insights into novel species of phycosphere bacteria and their ecological niches for an improved understanding of the macroalgal phycosphere microbiome. Video Abstract.
Asunto(s)
Laminaria , Microbiota , Rhodophyta , Algas Marinas , Ulva , Algas Marinas/microbiología , Ulva/genética , Ulva/microbiología , Laminaria/genética , ARN Ribosómico 16S/genética , Bacterias , Rhodophyta/genética , Microbiota/genéticaRESUMEN
BACKGROUND: Polycyclic triterpenoids (PTs) are common in plants, and have attracted considerable interest due to their remarkable biological activities. Currently, engineering the ergosterol synthesis pathway in Saccharomyces cerevisiae is a safe and cost-competitive way to produce triterpenoids. However, the strict regulation of ERG1 involved in the epoxidation of squalene limits the triterpenoid production. RESULTS: In this study, we found that the decrease in ERG7 protein level could dramatically boost the epoxidation of squalene by improving the protein stability of ERG1. We next explored the potential factors that affected the degradation process of ERG1 and confirmed that ERG7 was involved in the degradation process of ERG1. Subsequently, expression of four different triterpene cyclases utilizing either 2,3-oxidosqualene or 2,3:22,23-dioxidosqualene as the substrate in ERG7-degraded strains showed that the degradation of ERG7 to prompt the epoxidation of squalene could significantly increase triterpenoid production. To better display the potential of the strategy, we increased the supply of 2,3-oxidosqualene, optimized flux distribution between ergosterol synthesis pathway and ß-amyrin synthesis pathway, and modified the GAL-regulation system to separate the growth stage from the production stage. The best-performing strain ultimately produced 4216.6 ± 68.4 mg/L of ß-amyrin in a two-stage fed-fermentation (a 47-fold improvement over the initial strain). CONCLUSIONS: This study showed that deregulation of the native restriction in ergosterol pathway was an effective strategy to increase triterpenoid production in yeast, which provided a new insight into triterpenoids biosynthesis.
RESUMEN
l-homoserine, a nonprotein amino acid, is used to synthesize many active substances in the industry. Here, to develop a robust l-homoserine-producing strain, Escherichia coli W3110 was used as a chassis to be engineered. Based on a previous construct with blocked competing routes for l-homoserine synthesis, five genes were overexpressed by promoter replacement strategy to increase the l-homoserine production, including enhancement of precursors for l-homoserine synthesis (ppc, thrA, and asd), reinforcement of the NADPH supply (pntAB) and efflux transporters (rhtA) to improve the l-homoserine production. However, the plasmid losing was to blame for the wildly fluctuating fermentation performance of engineered strains, ranging between 2.1 and 6.2 g/L. Then, a hok/sok toxin/antitoxin system was introduced into the free plasmid expression cassette to maintain the genetic stability of the episomal plasmid; consequently, the plasmid-losing rate sharply decreased, resulting in the engineered strain SHL17, which exhibited excellent stability in l-homoserine production, with 6.3 g/L in shake flasks and 44.4 g/L in a 5-L fermenter without antibiotic addition. This work verified the effective use of the hok/sok toxin/antitoxin system combined with promoter engineering to improve the genetic stability of E. coli episomal plasmids without antibiotics.
Asunto(s)
Antitoxinas , Proteínas de Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Homoserina/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Antibacterianos/metabolismo , Plásmidos/genética , Antitoxinas/genética , Antitoxinas/metabolismo , Ingeniería Metabólica/métodosRESUMEN
Human epidermal growth factor (hEGF) has multiple biological functions, such as promoting cell proliferation, differentiation, and migration. In addition, it is a very expensive polypeptide with attractive market prospects. However, the production of hEGF needs for high cost to manufacture polypeptide demands reinvestigations of process conditions so as to enhance economic benefits. Improving the expression of soluble hEGF is the fundamental method to reduce the cost. In this study, a non-extracellular engineered strain of expressed hEGF was constructed, using plasmid pET-22b(+) in Escherichia coli. Preliminary fermentation and high cell density cultivation were carried out in shake flasks and in a 5 L bioreactor, respectively. A high yield of 98 ± 10 mg/L of soluble hEGF and a dry cell weight (DCW) of 6.98 ± 0.3 g/L were achieved in shake flasks. Then, fermentation conditions were optimized for large-scale production, while taking into consideration the expensive equipment required for cooling and conforming to industrial standards. A yield of 285 ± 10 mg/L of soluble hEGF, a final cell density of 57.4 ± 2 g/L DCW (OD600 141.1 ± 4.9), and hEGF productivity of 14.3 mg/L/h were obtained using a bioreactor at 32 °C for 20 h. The production method developed in this study for the biosynthesis of soluble hEGF is efficient and inexpensive.
Asunto(s)
Factor de Crecimiento Epidérmico , Escherichia coli , Humanos , Escherichia coli/metabolismo , Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Epidérmico/metabolismo , Plásmidos , Reactores Biológicos , FermentaciónRESUMEN
Rodents' lifestyles vary in different environments, and to adapt to various lifestyles specific digestion strategies have been developed. Among these strategies, the morphology of the digestive tracts and the gut microbiota are considered to play the most important roles in such adaptations. However, how subterranean rodents adapt to extreme environments through regulating gut microbial diversity and morphology of the digestive tract has yet to be fully studied. Here, we conducted the comparisons of the gastrointestinal morphology, food intake, food assimilation, food digestibility and gut microbiota of plateau zokor Eospalax baileyi in Qinghai-Tibet Plateau and laboratory rats Rattus norvegicus to further understand the survival strategy in a typical subterranean rodent species endemic to the Qinghai-Tibet Plateau. Our results revealed that plateau zokor evolved an efficient foraging strategy with low food intake, high food digestibility, and ultimately achieved a similar amount of food assimilation to laboratory rats. The length and weight of the digestive tract of the plateau zokor was significantly higher than the laboratory rat. Particularly, the weight and length of the large intestine and cecum in plateau zokor is three times greater than that of the laboratory rat. Microbiome analysis showed that genus (i.e., Prevotella, Oscillospira, CF231, Ruminococcus and Bacteroides), which are usually associated with cellulose degradation, were significantly enriched in laboratory rats, compared to plateau zokor. However, prediction of metagenomic function revealed that both plateau zokor and laboratory rats shared the same functions in carbohydrate metabolism and energy metabolism. The higher digestibility of crude fiber in plateau zokor was mainly driven by the sizes of cecum and cecum tract, as well as those gut microbiota which associated with cellulose degradation. Altogether, our results highlight that both gut microbiota and the morphology of the digestive tract are vital to the digestion in wild rodents.
RESUMEN
Genomic integration of genes and pathway-sized DNA cassettes is often an indispensable way to construct robust and productive microbial cell factories. For some uncommon microbial hosts, such as Mycolicibacterium and Mycobacterium species, however, it is a challenge. Here, we present a multiplexed integrase-assisted site-specific recombination (miSSR) method to precisely and iteratively integrate genes/pathways with controllable copies in the chromosomes of Mycolicibacteria for the purpose of developing cell factories. First, a single-step multi-copy integration method was established in M. neoaurum by a combination application of mycobacteriophage L5 integrase and two-step allelic exchange strategy, the efficiencies of which were â¼100% for no more than three-copy integration events and decreased sharply to â¼20% for five-copy integration events. Second, the R4, Bxb1 and ΦC31 bacteriophage Att/Int systems were selected to extend the available integration toolbox for multiplexed gene integration events. Third, a reconstructed mycolicibacterial Xer recombinases (Xer-cise) system was employed to recycle the selection marker of gene recombination to facilitate the iterative gene manipulation. As a proof of concept, the biosynthetic pathway of ergothioneine (EGT) in Mycolicibacterium neoaurum ATCC 25795 was achieved by remodeling its metabolic pathway with a miSSR system. With six copies of the biosynthetic gene clusters (BGCs) of EGT and pentose phosphate isomerase (PRT), the titer of EGT in the resulting strain in a 30 mL shake flask within 5 days was enhanced to 66 mg/L, which was 3.77 times of that in the wild strain. The improvements indicated that the miSSR system was an effective, flexible, and convenient tool to engineer the genomes of Mycolicibacteria as well as other strains in the Mycobacteriaceae due to their proximate evolutionary relationships.
RESUMEN
To investigate the responses of key enzymes involved in steroidal saponin biosynthesis of Dioscorea zingiberensis to low phosphorus stress, we designed three treatments of severe phosphorus stress, moderate phosphorus stress, and normal phosphorus level. The D. zingiberensis plants were collected at the early, middle, and late stages of treatment. The content of total steroidal saponins in different tissues of D. zingiberensis was determined by spectrophotometry for the identification of the critical stage in response to low phosphorus stress. BGI 500 sequencing platform was employed to obtain the transcript information of D. zingiberensis samples at the critical stage of low phosphorus stress, and then a transcriptome library was constructed. The correlation between the expression of genes involved in steroidal saponin biosynthesis and the content of total steroidal saponins was analyzed for the screening of the key enzyme genes in response to low phosphorus stress. Further, the expression patterns of these genes were analyzed by real-time fluorescence PCR(qRT-PCR). The content of total steroidal saponins in D. zingiberensis had obvious tissue specificity under low phosphorus stress, and the early stage of stress was particularly important for D. zingiberensis to respond to low phosphorus stress. A total of 101 593 unigenes were obtained by transcriptome sequencing, of which 77.35% were annotated in NT, NR, SwissProt, KOG, GO, and KEGG. A total of 256 transcripts of known key enzyme genes in the biosynthetic pathway of steroidal saponins were identified. The expression levels of 69 transcripts encoding 18 catalytic enzymes were significantly correlated with the content of total steroidal saponins. The qRT-PCR results showed that several key enzyme genes presented different expression patterns in four tissues under low phosphorus stress. The results indicated that the content of total steroidal saponins and the expression of key enzyme genes regulating steroidal saponin biosynthesis in D. zingensis changed under low phosphorus stress. This study provides the biological information for elucidating the molecular mechanism of steroidal saponin biosynthesis in D. zingensis exposed to low phosphorus stress.