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Considered a "Generally Recognized As Safe" (GRAS) bacterium, the plant growth-promoting rhizobacterium Paenibacillus has been widely applied in: agriculture, medicine, industry, and environmental remediation. Paenibacillus species not only accelerate plant growth and degrade toxic substances in wastewater and soil but also produce industrially-relevant enzymes and antimicrobial peptides. Due to a lack of genetic manipulation tools and methods, exploitation of the bioresources of naturally isolated Paenibacillus species has long been limited. Genetic manipulation tools and methods continue to improve in Paenibacillus, such as shuttle plasmids, promoters, and genetic tools of CRISPR. Furthermore, genetic transformation systems develop gradually, including: penicillin-mediated transformation, electroporation, and magnesium amino acid-mediated transformation. As genetic manipulation methods of homologous recombination and CRISPR-mediated editing system have developed gradually, Paenibacillus has come to be regarded as a promising microbial chassis for biomanufacturing, expanding its application scope, such as: industrial enzymes, bioremediation and bioadsorption, surfactants, and antibacterial agents. In this review, we describe the applications of Paenibacillus bioproducts, and then discuss recent advances and future challenges in the development of genetic manipulation systems in this genus. This work highlights the potential of Paenibacillus as a new microbial chassis for mining bioresources.
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ß-alanine has been used in food and pharmaceutical industries. Although Escherichia coli Nissle 1917 (EcN) is generally considered safe and engineered as living therapeutics, engineering EcN for producing industrial metabolites has rarely been explored. Here, by protein and metabolic engineering, EcN was engineered for producing ß-alanine from glucose. First, an aspartate-α-decarboxylase variant ADCK43Y with improved activity was identified and over-expressed in EcN, promoting the titer of ß-alanine from an undetectable level to 0.46 g/L. Second, directing the metabolic flux towards L-aspartate increased the titer of ß-alanine to 0.92 g/L. Third, the yield of ß-alanine was elevated to 1.19 g/L by blocking conversion of phosphoenolpyruvate to pyruvate, and further increased to 2.14 g/L through optimizing culture medium. Finally, the engineered EcN produced 11.9 g/L ß-alanine in fed-batch fermentation. Our work not only shows the potential of EcN as a valuable industrial platform, but also facilitates production of ß-alanine via fermentation. KEY POINTS: ⢠Escherichia coli Nissle 1917 (EcN) was engineered as a ß-alanine producing cell factory ⢠Identification of a decarboxylase variant ADCK43Y with improved activity ⢠Directing the metabolic flux to L-ASP and expressing ADCK43Y elevated the titer of ß-alanine to 11.9 g/L.
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Ingeniería Metabólica , Probióticos , Escherichia coli/metabolismo , beta-Alanina/metabolismo , FermentaciónRESUMEN
Fat-soluble vitamins are vitamins that are insoluble in water, soluble in fat, and organic solvents; they are found in minute amount in various foods. Fat-soluble vitamins, including vitamins A, D, E, and K, have been widely used in food, cosmetics, health care products, and pharmaceutical industries. Fat-soluble vitamins are currently produced via biological and chemical synthesis. In recent years, fat-soluble vitamin production by biotechnological routes has been regarded as a very promising approach. Based on biosynthetic pathways, considerable advances of α-tocopherol and ß-carotenes have been achieved in transgenic plants and microalgae. Microbial fermentation, as an alternative method for the production of vitamin K and ß-carotenes, is attracting considerable attention because it is an environment friendly process. In this review, we address the function and applications of fat-soluble vitamins, and an overview of current developments in the production of fat-soluble vitamins in transgenic plants, microalgae, and microorganisms. We focus on the metabolic and process engineering strategies for improving production of fat-soluble vitamins, and we hope this review can be useful for the people who are interested in the production of fat-soluble vitamins by biotechnological routes.
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Grasas/química , Ingeniería Metabólica , Vitaminas/biosíntesis , Vías Biosintéticas , Biotecnología , Fermentación , Solubilidad , Vitamina A/biosíntesis , Vitamina D/biosíntesis , Vitamina E/biosíntesis , Vitamina K/biosíntesisRESUMEN
Low-molecular-weight hyaluronan (LMW-HA) has attracted much attention because of its many potential applications. Here, we efficiently produced specific LMW-HAs from sucrose in Bacillus subtilis. By coexpressing the identified committed genes (tuaD, gtaB, glmU, glmM, and glmS) and downregulating the glycolytic pathway, HA production was significantly increased from 1.01gL(-1) to 3.16gL(-1), with a molecular weight range of 1.40×10(6)-1.83×10(6)Da. When leech hyaluronidase was actively expressed after N-terminal engineering (1.62×10(6)UmL(-1)), the production of HA was substantially increased from 5.96gL(-1) to 19.38gL(-1). The level of hyaluronidase was rationally regulated with a ribosome-binding site engineering strategy, allowing the production of LMW-HAs with a molecular weight range of 2.20×10(3)-1.42×10(6)Da. Our results confirm that this strategy for the controllable expression of hyaluronidase, together with the optimization of the HA synthetic pathway, effectively produces specific LMW-HAs, and could also be used to produce other LMW polysaccharides.
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Bacillus subtilis , Ácido Hialurónico , Ingeniería Metabólica , Animales , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Ácido Hialurónico/biosíntesis , Ácido Hialurónico/genética , Hialuronoglucosaminidasa/biosíntesis , Hialuronoglucosaminidasa/genética , Sanguijuelas/enzimología , Sanguijuelas/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genéticaRESUMEN
The CRISPR-based regulation tools enable fine-tuning of gene transcription, showing potential in areas of biomanufacturing and live therapeutics. However, the cell toxicity and PAM specificity of existing CRISPR-based regulation systems limit their broad application. The development of new and less-toxic CRISPR-controlled expression systems remains highly desirable for expanding the application scope of CRISPR-based tools. Here, we reconstituted the type I CRISPR-Cas system from Escherichia coli to finely tune gene expression in Bacillus subtilis. Through engineering the 5' untranslated region (UTR) of mRNAs of cas genes, we remarkably improved the efficacy of the type I CRISPRi system. The improved type I CRISPRi system was applied in engineering the D-pantothenic acid (DPA)-producing B. subtilis, which was generated by strengthening the metabolic flux toward ß-alanine and (R)-pantoate via enhancing expression of key enzymes at both transcriptional and translational levels. Through controlling the expression of pdhA with the CRISPRi system for fine-tuning the metabolic flux toward DPA and the TCA cycle, we elevated the DPA titer to 0.88 g/L in shake flasks and 12.81 g/L in fed-batch fermentations without the addition of the precursor ß-alanine. The type I CRISPRi system and the strategy for fine-tuning metabolic flux reported here not only enrich the CRISPR toolbox in B. subtilis and facilitate DPA production through microbial fermentation but also provide a paradigm for programming important organisms to produce value-added chemicals with cheap raw materials.
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In response to a high concentration of glucose, Bacillus subtilis, a microbial chassis for producing many industrial metabolites, rapidly takes up glucose using the phosphotransferase system (PTS), leading to overflow metabolism, a common phenomenon observed in many bacteria. Although overflow metabolism affects cell growth and reduces the production of many metabolites, effective strategies that reduce overflow metabolism while maintaining normal cell growth remain to be developed. Here, we used a quorum sensing (QS)-mediated circuit to tune the glucose uptake rate and thereby relieve overflow metabolism in an engineered B. subtilis for producing d-pantothenic acid (DPA). A low-efficiency non-PTS system was used for glucose uptake at the early growth stages to avoid a rapid glycolytic flux, while an efficient PTS system, which was activated by a QS circuit, was automatically activated at the late growth stages after surpassing a threshold cell density. This strategy was successfully applied as a modular metabolic engineering process for the high production of DPA. By enhancing the translation levels of key enzymes (3-methyl-2-oxobutanoate hydroxymethytransferase, pantothenate synthetase, aspartate 1-decarboxylase proenzyme, 2-dehydropantoate 2-reductase, dihydroxy-acid dehydratase, and acetolactate synthase) with engineered 5'-untranslated regions (UTRs) of mRNAs, the metabolic flux was promoted in the direction of DPA production, elevating the yield of DPA to 5.11 g/L in shake flasks. Finally, the engineered B. subtilis produced 21.52 g/L of DPA in fed-batch fermentations. Our work not only revealed a new strategy for reducing overflow metabolism by adjusting the glucose uptake rate in combination with promoting the translation of key metabolic enzymes through engineering the 5'-UTR of mRNAs but also showed its power in promoting the bioproduction of DPA in B. subtilis, exhibiting promising application prospects.
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Bacillus subtilis , Ácido Pantoténico , Bacillus subtilis/metabolismo , Ácido Pantoténico/metabolismo , Percepción de Quorum , Metabolismo de los Hidratos de Carbono , Glucosa/metabolismo , Ingeniería MetabólicaRESUMEN
Considered a "Generally Recognized As Safe" (GRAS) bacterium, the plant growth-promoting rhizobacterium Paenibacillus polymyxa has been widely applied in agriculture and animal husbandry. It also produces valuable compounds that are used in medicine and industry. Our previous work showed the presence of restriction modification (RM) system in P. polymyxa ATCC 842. Here, we further analyzed its genome and methylome by using SMRT sequencing, which revealed the presence of a larger number of genes, as well as a plasmid documented as a genomic region in a previous report. A number of mobile genetic elements (MGEs), including 78 insertion sequences, six genomic islands, and six prophages, were identified in the genome. A putative lysozyme-encoding gene from prophage P6 was shown to express lysin which caused cell lysis. Analysis of the methylome and genome uncovered a pair of reverse-complementary DNA methylation motifs which were widespread in the genome, as well as genes potentially encoding their cognate type I restriction-modification system PpoAI. Further genetic analysis confirmed the function of PpoAI as a RM system in modifying and restricting DNA. The average frequency of the DNA methylation motifs in MGEs was lower than that in the genome, implicating a role of PpoAI in restricting MGEs during genomic evolution of P. polymyxa. Finally, comparative analysis of R, M, and S subunits of PpoAI showed that homologs of the PpoAI system were widely distributed in species belonging to other classes of Firmicute, implicating a role of the ancestor of PpoAI in the genomic evolution of species beyond Paenibacillus.
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Menaquinone-4 (MK-4) plays a significant role in bone health and cardiovascular therapy. Although many strategies have been adopted to increase the yield of MK-4 in Bacillus subtilis 168, the effectiveness of MK-4 is still low due to the inherent limitations of metabolic pathways. However, dynamic regulation based on quorum sensing (QS) has been extensively applied as a fundamental tool for fine-tuning gene expression in reaction to changes in cell density without adding expensive inducers. Nevertheless, in most reports, QS systems depend on down-regulated expression rather than up-regulated expression, which greatly limit their potential as molecular switches to control metabolic flux. To address this challenge, a modular PhrQ-RapQ-ComA QS system is developed based on promoter PA11, which is up-regulated by phosphorylated ComA (ComA-P). In this paper, firstly we analyzed the ComA-based gene expression regulation system in Bacillus subtilis 168. We constructed a promoter library of diï¬ ;erent abilities, selected best promoters from a library, and performed mutation screening on the selected promoters. Furthermore, we constructed a PhrQ-RapQ-ComA QS system to dynamically control the synthesis of MK-4 in B. subtilis 168. Cell growth and eï¬cient synthesis of the target product can be dynamically balanced by the QS system. Our dynamic adjustment approach increased the yield of MK-4 in shake flask from 120.1 ± 0.6 to 178.9 ± 2.8 mg/L, and reached 217 ± 4.1 mg/L in a 3-L bioreactor, which verified the effectiveness of this strategy. In summary, PhrQ-RapQ-ComA QS system can realize dynamic pathway regulation in B. subtilis 168, which can be stretched to a great deal of microorganisms to fine-tune gene expression and enhance the production of metabolites.
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Bacillus subtilis , Percepción de Quorum , Bacillus subtilis/genética , Regulación Bacteriana de la Expresión Génica , Ingeniería Metabólica , Vitamina K 2/análogos & derivadosRESUMEN
Menaquinone-4 (MK-4), one form of vitamin K, plays an important role in cardiovascular and bone health. Menaquinone-4 (MK-4) is a valuable vitamin K2 that is diï¬cult to synthesize organically, and now is mainly produced by microbial fermentation. Herein we significantly improved the synthesis efficiency of MK-4 by combinatorial pathway engineering in Bacillus subtilis 168, a model industrial strain widely used for production of nutraceuticals. The metabolic networks related to MK-4 synthesis include four modules, namely, MK-4 biosynthesis module, methylerythritol phosphate (MEP) module, mevalonate-dependent (MVA) isoprenoid module, and menaquinone module. Overexpression of menA, menG, and crtE genes from Synechocystis sp. PCC 6803 in MK-4 synthesis module with strong constitutive promoter P43 resulted in 8.1 ± 0.2 mg/L of MK-4 (No MK-4 was detected in the wild-type B. subtilis 168). MK-4 titer was further increased by 3.8-fold to 31.53 ± 0.95 mg/L by knockout of hepT gene, which catalyzes the conversion of Farnesyl diphosphate to Heptaprenyl diphosphate. In addition, simultaneous overexpression of dxs, dxr, and ispD-ispF genes in MEP module with strong promoter P43 increased the titer of MK-4 to 78.1 ± 1.6 mg/L. Moreover, expression of the heterogeneous MVA module genes (mvaK1, mvaK2, mvaD, mvaS, and mvaA) resulted in 90.1 ± 1.7 mg/L of MK-4. Finally, in order to further convert the enhanced carbon metabolism flux to MK-4, simultaneous overexpression of the genes crtE, menA, and menG in menaquinone pathway with strong promoter P43 increased the titer of MK-4 to 120.1 ± 0.6 mg/L in shake flask and 145 ± 2.8 mg/L in a 3-L fed-batch bioreactor. Herein the engineered B. subtilis strain may be used for the industrial production of MK-4 in the future.
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Bacillus subtilis/metabolismo , Vitamina K 2/análogos & derivados , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Reactores Biológicos , Eritritol/análogos & derivados , Eritritol/metabolismo , Fermentación , Hemiterpenos/metabolismo , Ingeniería Metabólica , Redes y Vías Metabólicas , Ácido Mevalónico/metabolismo , Compuestos Organofosforados/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fosfatos de Azúcar/metabolismo , Synechocystis/genética , Vitamina K 2/metabolismoRESUMEN
Hyaluronan oligosaccharides (o-HAs), especially saturated o-HAs, have attracted intensive attention due to their potential applications in medical treatments. In this study, the hydrolysis process of leech hyaluronidase (LHase) towards the hyaluronan was investigated by HPLC and HPLC/ESI-MS. The proportions of hyaluronan tetrasaccharide (HA4) with hexasaccharide (HA6), end products, were illustrated to have a relationship with the amount of LHase. Higher yield of HA4 was achieved with higher activity of LHase. After optimisation of the packing resin and operation parameters (balanced pH, elution concentration, elution volume and elution flow rate), the highly pure HA4 and HA6 were efficiently separated and prepared by combining ion exchange Q-Sepharose Fast Flow and size exclusion column chromatography. Compared with o-HAs (average Mr of 4000 Da), HA4 and HA6 were demonstrated to show higher activity for promoting angiogenesis, which was similar with the corresponding HA4 and HA6 produced by bovine testicular hyaluronidase. The pure HA4 and HA6 that prepared from LHase will attract intensive studies and be used in potential applications in near future.
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Inductores de la Angiogénesis/química , Inductores de la Angiogénesis/farmacología , Ácido Hialurónico/química , Ácido Hialurónico/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Oligosacáridos/química , Oligosacáridos/farmacología , Inductores de la Angiogénesis/metabolismo , Animales , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Femenino , Ácido Hialurónico/metabolismo , Hialuronoglucosaminidasa/metabolismo , Hidrólisis , Sanguijuelas/enzimología , Ratones , Oligosacáridos/metabolismo , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Chondroitin and heparosan, important polysaccharides and key precursors of chondroitin sulfate and heparin/heparan sulfate, have drawn much attention due to their wide applications in many aspects. In this study, we designed two independent synthetic pathways of chondroitin and heparosan in food-grade Bacillus subtilis, integrating critical synthases genes derived from Escherichia coli into B. subtilis genome. By RT-PCR analysis, we confirmed that synthases genes transcripted an integral mRNA chain, suggesting co-expression. In shaken flask, chondroitin and heparosan were produced at a level of 1.83gL(-1) and 1.71gL(-1), respectively. Since B. subtilis endogenous tuaD gene encodes the limiting factor of biosynthesis, overexpressing tuaD resulted in enhanced chondroitin and heparosan titers, namely 2.54gL(-1) and 2.65gL(-1). Moreover, production reached the highest peaks of 5.22gL(-1) and 5.82gL(-1) in 3-L fed-batch fermentation, respectively, allowed to double the production that in shaken flask. The weight-average molecular weight of chondroitin and heparosan from B. subtilis E168C/pP43-D and E168H/pP43-D were 114.07 and 67.70kDa, respectively. This work provided alternative safer synthetic pathways for metabolic engineering of chondroitin and heparosan in B. subtilis and a useful approach for enhancing production, which can be optimized for further improvement.
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Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Condroitín/biosíntesis , Disacáridos/biosíntesis , Ingeniería Metabólica , Proteínas Bacterianas/genética , Técnicas de Cultivo Celular por Lotes , Condroitín/química , ADN Recombinante/genética , Disacáridos/química , Fermentación , Peso Molecular , Regulación hacia ArribaRESUMEN
High-molecular-mass hyaluronan (HA) was controllably depolymerized in pure aqueous solution with recombinant leech hyaluronidase (HAase). The HAase concentration per unit HA and hydrolysis time played important roles in molecular mass distribution. By modulating the concentrations of HAase and controlling the hydrolysis time, any molar-mass-defined HA oligomers could be efficiently and specifically produced on a large scale (40 g/L), such as HA oligosaccharides with weight-average molar mass of 4000, 10,000, and 30,000Da and end hydrolysates containing only HA6 and HA4. High performance liquid chromatography-size exclusion chromatography, polyacrylamide gel electrophoresis, capillary zone electrophoresis, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry confirmed low polydispersity of the produced molar-mass-defined HA oligosaccharides. Therefore, large-scale production of defined HA oligosaccharides with narrow molecular mass distribution will significantly promote progress in related research and its potential applications.