RESUMEN

Acid stress induced by the accumulation of organic acids during the fermentation of propionibacteria is a severe limitation in the microbial production of propionic acid (PA). To enhance the acid resistance of strains, the tolerance mechanisms of cells must first be understood. In this study, comparative genomic and transcriptomic analyses were conducted on wild-type and acid-tolerant Propionibacterium acidipropionici to reveal the microbial response of cells to acid stress during fermentation. Combined with the results of previous proteomic and metabolomic studies, several potential acid-resistance mechanisms of P. acidipropionici were analyzed. Energy metabolism and transporter activity of cells were regulated to maintain pH homeostasis by balancing transmembrane transport of protons and ions; redundant protons were eliminated by enhancing the metabolism of certain amino acids for a relatively stable intracellular microenvironment; and protective mechanism of macromolecules were also induced to repair damage to proteins and DNA by acids. Transcriptomic data indicated that the synthesis of acetate and lactate were undesirable in the acid-resistant mutant, the expression of which was 2.21-fold downregulated. In addition, metabolomic data suggested that the accumulation of lactic acid and acetic acid reduced the carbon flow to PA and led to a decrease in pH. On this basis, we propose a metabolic engineering strategy to regulate the synthesis of lactic acid and acetic acid that will reduce by-products significantly and increase the PA yield by 12.2% to 10.31 ± 0.84 g/g DCW. Results of this study provide valuable guidance to understand the response of bacteria to acid stress and to construct microbial cell factories to produce organic acids by combining systems biology technologies with synthetic biology tools.

Asunto(s)

Perfilación de la Expresión Génica/métodos , Genómica/métodos , Ingeniería Metabólica/métodos , Propionatos/metabolismo , Propionibacterium , Ácidos , Adaptación Biológica/genética , Propionibacterium/genética , Propionibacterium/metabolismo , Propionibacterium/fisiología

RESUMEN

Microbial production of propionic acid (PA), an important chemical building block used as a preservative and chemical intermediate, has gained increasing attention for its environmental friendliness over traditional petrochemical processes. In previous studies, we constructed a shuttle vector as a useful tool for engineering Propionibacterium jensenii, a potential candidate for efficient PA synthesis. In this study, we identified the key metabolites for PA synthesis in P. jensenii by examining the influence of metabolic intermediate addition on PA synthesis with glycerol as a carbon source under anaerobic conditions. We also further improved PA production via the overexpression of the identified corresponding enzymes, namely, glycerol dehydrogenase (GDH), malate dehydrogenase (MDH), and fumarate hydratase (FUM). Compared to those in wild-type P. jensenii, the activities of these enzymes in the engineered strains were 2.91- ± 0.17- to 8.12- ± 0.37-fold higher. The transcription levels of the corresponding enzymes in the engineered strains were 2.85- ± 0.19- to 8.07- ± 0.63-fold higher than those in the wild type. The coexpression of GDH and MDH increased the PA titer from 26.95 ± 1.21 g/liter in wild-type P. jensenii to 39.43 ± 1.90 g/liter in the engineered strains. This study identified the key metabolic nodes limiting PA overproduction in P. jensenii and further improved PA titers via the coexpression of GDH and MDH, making the engineered P. jensenii strain a potential industrial producer of PA.

Asunto(s)

Klebsiella pneumoniae/enzimología , Malato Deshidrogenasa/metabolismo , Ingeniería Metabólica , Propionatos/metabolismo , Propionibacterium/metabolismo , Proteínas Recombinantes/metabolismo , Deshidrogenasas del Alcohol de Azúcar/metabolismo , Anaerobiosis , Carbono/metabolismo , Fumarato Hidratasa/genética , Fumarato Hidratasa/metabolismo , Expresión Génica , Perfilación de la Expresión Génica , Vectores Genéticos , Glicerol/metabolismo , Klebsiella pneumoniae/genética , Malato Deshidrogenasa/genética , Propionibacterium/genética , Proteínas Recombinantes/genética , Deshidrogenasas del Alcohol de Azúcar/genética , Transcripción Genética

RESUMEN

Propionic acid (PA) is an important platform chemical in the food, agriculture, and pharmaceutical industries and is mainly biosynthesized by propionibacteria. Acid tolerance in PA-producing strains is crucial. In previous work, we investigated the acid tolerance mechanism of Propionibacterium acidipropionici at microenvironmental levels by analyzing physiological changes in the parental strain and three PA-tolerant mutants obtained by genome shuffling. However, the molecular mechanism of PA tolerance in P. acidipropionici remained unclear. Here, we performed a comparative proteomics study of P. acidipropionici CGMCC 1.2230 and the acid-tolerant mutant P. acidipropionici WSH1105; MALDI-TOF/MS identified 24 proteins that significantly differed between the parental and shuffled strains. The differentially expressed proteins were mainly categorized as key components of crucial biological processes and the acid stress response. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was used to confirm differential expression of nine key proteins. Overexpression of the secretory protein glyceraldehyde-3-phosphate dehydrogenase and ATP synthase subunit α in Escherichia coli BL21 improved PA and acetic acid tolerance; overexpression of NADH dehydrogenase and methylmalonyl-CoA epimerase improved PA tolerance. These results provide new insights into the acid tolerance of P. acidipropionici and will facilitate the development of PA production through fermentation by propionibacteria.

Asunto(s)

Proteínas Bacterianas/genética , Barajamiento de ADN/métodos , Regulación Bacteriana de la Expresión Génica , Propionatos/metabolismo , Propionibacterium/genética , Proteómica , Ácido Acético/metabolismo , Adaptación Fisiológica , Proteínas Bacterianas/metabolismo , ATPasas de Translocación de Protón Bacterianas/genética , ATPasas de Translocación de Protón Bacterianas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fermentación , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/genética , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/metabolismo , NADH Deshidrogenasa/genética , NADH Deshidrogenasa/metabolismo , Propionibacterium/metabolismo , Racemasas y Epimerasas/genética , Racemasas y Epimerasas/metabolismo , Estrés Fisiológico , Transgenes

RESUMEN

In this study, we fused six self-assembling amphipathic peptides (SAPs) with cyclodextrin glycosyltransferase (CGTase) from Paenibacillus macerans to catalyze 2-O-D-glucopyranosyl-L-ascorbic acid (AA-2G) production with cheap substrates, including maltose, maltodextrin, and soluble starch as glycosyl donors. The results showed that two fusion enzymes, SAP5-CGTase and SAP6-CGTase, increased AA-2G yields to 2.33- and 3.36-fold that of wild-type CGTase when soluble starch was used as a substrate. The cyclization activities of these enzymes decreased, while disproportionation activities increased. Enzymatic characterization of the two fusion enzymes was performed, and kinetics analysis of AA-2G synthesis confirmed the enhanced soluble starch specificity of SAP5-CGTase and SAP6-CGTase compared to that in the wild-type CGTase. As revealed by structure modeling of the fusion and wild-type CGTases, enhanced substrate-binding capacity may result from the increased number of hydrogen bonds present after fusion. This study demonstrates an effective protein fusion approach to improving the substrate specificity of CGTase for AA-2G synthesis. Fusion enzymes, especially SAP6-CGTase, are promising starting points for further development through protein engineering.

Asunto(s)

Ácido Ascórbico/análogos & derivados , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Oligopéptidos/metabolismo , Paenibacillus/enzimología , Almidón/metabolismo , Ácido Ascórbico/biosíntesis , Proteínas Bacterianas/química , Biocatálisis , Glucosiltransferasas/química , Cinética , Oligopéptidos/genética , Paenibacillus/genética , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Especificidad por Sustrato

RESUMEN

In previous work, we constructed a recombinant Bacillus subtilis strain for microbial production of N-acetylglucosamine (GlcNAc), which has applications in nutraceuticals and pharmaceuticals. In this work, we improve GlcNAc production through modular engineering of B. subtilis. Specifically, the GlcNAc synthesis-related metabolic network in B. subtilis was divided into three modules-GlcNAc synthesis, glycolysis, and peptidoglycan synthesis. First, two-promoter systems with different promoter types and strengths were used for combinatorial assembly of expression cassettes of glmS (encoding GlcN-6-phosphate synthase) and GNA1 (encoding GlcNAc-6-phosphate N-acetyltransferase) at transcriptional levels in the GlcNAc synthesis module, resulting in a 32.4% increase in GlcNAc titer (from 1.85g/L to 2.45g/L) in shake flasks. In addition, lactate and acetate synthesis were blocked by knockout of ldh (encoding lactate dehydrogenase) and pta (encoding phosphotransacetylase), leading to a 44.9% increase in GlcNAc production (from 2.45g/L to 3.55g/L) in shake flasks. Then, various strengths of the glycolysis and peptidoglycan synthesis modules were constructed by repressing the expression of pfk (encoding 6-phosphofructokinase) and glmM (encoding phosphoglucosamine mutase) via the expression of various combinations of synthetic small regulatory RNAs and Hfq protein. Next, GlcNAc, glycolysis, and peptidoglycan synthesis modules with various strengths were assembled and optimized via a module engineering approach, and the GlcNAc titer was improved to 8.30g/L from 3.55g/L in shake flasks. Finally, the GlcNAc titer was further increased to 31.65g/L, which was 3.8-fold that in the shake flask, in a 3-L fed-batch bioreactor. This work significantly enhanced GlcNAc production through modular pathway engineering of B. subtilis, and the engineering strategies used herein may be useful for the construction of versatile B. subtilis cell factories for the production of other industrially important chemicals.

Asunto(s)

Acetilglucosamina , Acetiltransferasas , Bacillus subtilis , Ingeniería Metabólica/métodos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Acetilglucosamina/biosíntesis , Acetilglucosamina/genética , Acetiltransferasas/biosíntesis , Acetiltransferasas/genética , Bacillus subtilis/enzimología , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Técnicas de Silenciamiento del Gen , L-Lactato Deshidrogenasa/genética , L-Lactato Deshidrogenasa/metabolismo , Fosfato Acetiltransferasa/genética , Fosfato Acetiltransferasa/metabolismo , Fosfofructoquinasa-1/genética , Fosfofructoquinasa-1/metabolismo , Fosfoglucomutasa/genética , Fosfoglucomutasa/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/biosíntesis , Proteínas de Saccharomyces cerevisiae/genética , Transcripción Genética/genética

RESUMEN

Cyclodextrin glycosyltransferase (CGTase) is an important enzyme with multiple functions, in particular the production of cyclodextrins. It is also widely applied in baking and carbohydrate glycosylation because it participates in various types of catalytic reactions. New applications are being found with novel CGTases being isolated from various organisms. Heterologous expression is performed for the overproduction of CGTases to meet the requirements of these applications. In addition, various directed evolution techniques have been applied to modify the molecular structure of CGTase for improved performance in industrial applications. In recent years, substantial progress has been made in the heterologous expression and molecular engineering of CGTases. In this review, we systematically summarize the heterologous expression strategies used for enhancing the production of CGTases. We also outline and discuss the molecular engineering approaches used to improve the production, secretion, and properties (e.g., product and substrate specificity, catalytic efficiency, and thermal stability) of CGTase.

Asunto(s)

Evolución Molecular Dirigida , Glucosiltransferasas , Ingeniería de Proteínas , Bacterias/enzimología , Bacterias/genética , Bacterias/metabolismo , Glucosiltransferasas/química , Glucosiltransferasas/genética , Glucosiltransferasas/aislamiento & purificación , Glucosiltransferasas/metabolismo , Proteínas Recombinantes

RESUMEN

The goal of this work was to develop an immobilized whole-cell biocatalytic process for the environment-friendly synthesis of α-ketoglutaric acid (α-KG) from l-glutamic acid. We compared the suitability of Escherichia coli and Bacillus subtilis strains overexpressing Proteus mirabilisl-amino acid deaminase (l-AAD) as potential biocatalysts. Although both recombinant strains were biocatalytically active, the performance of B. subtilis was superior to that of E. coli. With l-glutamic acid as the substrate, α-KG production levels by membranes isolated from B. subtilis and E. coli were 55.3±1.73 and 21.7±0.39µg/mg protein/min, respectively. The maximal conversion ratio of l-glutamic acid to α-KG was 31% (w/w) under the following optimal conditions: 15g/L l-glutamic acid, 20g/L whole-cell biocatalyst, 5mM MgCl2, 40°C, pH 8.0, and 24-h incubation. Immobilization of whole cells with alginate increased the recyclability by an average of 23.33% per cycle. This work established an efficient one-step biotransformation process for the production of α-KG using immobilized whole B. subtilis overexpressing P. mirabilisl-AAD. Compared with traditional multistep chemical synthesis, the biocatalytic process described here has the advantage of reducing environmental pollution and thus has great potential for the large-scale production of α-KG.

Asunto(s)

Proteínas Bacterianas/metabolismo , D-Aminoácido Oxidasa/metabolismo , Ácido Glutámico/metabolismo , Ácidos Cetoglutáricos/metabolismo , Proteus mirabilis/enzimología , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Biocatálisis , Células Inmovilizadas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica/genética , Proteínas Recombinantes/metabolismo

RESUMEN

High thermostability is required for alkaline α-amylases to maintain high catalytic activity under the harsh conditions used in textile production. In this study, we attempted to improve the thermostability of an alkaline α-amylase from Alkalimonas amylolytica through in silico rational design and systems engineering of disulfide bridges in the catalytic domain. Specifically, 7 residue pairs (P35-G426, Q107-G167, G116-Q120, A147-W160, G233-V265, A332-G370, and R436-M480) were chosen as engineering targets for disulfide bridge formation, and the respective residues were replaced with cysteines. Three single disulfide bridge mutants-P35C-G426C, G116C-Q120C, and R436C-M480C-of the 7 showed significantly enhanced thermostability. Combinational mutations were subsequently assessed, and the triple mutant P35C-G426C/G116C-Q120C/R436C-M480C showed a 6-fold increase in half-life at 60°C and a 5.2°C increase in melting temperature compared with the wild-type enzyme. Interestingly, other biochemical properties of this mutant also improved: the optimum temperature increased from 50°C to 55°C, the optimum pH shifted from 9.5 to 10.0, the stable pH range extended from 7.0 to 11.0 to 6.0 to 12.0, and the catalytic efficiency (kcat/Km) increased from 1.8 × 10(4) to 2.4 × 10(4) liters/g · min. The possible mechanism responsible for these improvements was explored through comparative analysis of the model structures of wild-type and mutant enzymes. The disulfide bridge engineering strategy used in this work may be applied to improve the thermostability of other industrial enzymes.

Asunto(s)

Dominio Catalítico , Cisteína/genética , Cisteína/metabolismo , Disulfuros/metabolismo , Gammaproteobacteria/enzimología , Ingeniería de Proteínas/métodos , alfa-Amilasas/metabolismo , Estabilidad de Enzimas , Gammaproteobacteria/genética , Temperatura , alfa-Amilasas/química , alfa-Amilasas/genética

RESUMEN

2-O-d-Glucopyranosyl-l-ascorbic acid (AA-2G), a stable l-ascorbic acid derivative, is usually synthesized by cyclodextrin glycosyltransferase (CGTase), which contains nine substrate-binding subsites (from +2 to -7). In this study, iterative saturation mutagenesis (ISM) was performed on the -6 subsite residues (Y167, G179, G180, and N193) in the CGTase from Paenibacillus macerans to improve its specificity for maltodextrin, which is a cheap and easily soluble glycosyl donor for AA-2G synthesis. Site saturation mutagenesis of four sites-Y167, G179, G180, and N193-was first performed and revealed that four mutants-Y167S, G179R, N193R, and G180R-produced AA-2G yields higher than those of other mutant and wild-type CGTases. ISM was then conducted with the best positive mutant as a template. Under optimal conditions, mutant Y167S/G179K/N193R/G180R produced the highest AA-2G titer of 2.12 g/liter, which was 84% higher than that (1.15 g/liter) produced by the wild-type CGTase. Kinetics analysis of AA-2G synthesis using mutant CGTases confirmed the enhanced maltodextrin specificity and showed that compared to the wild-type CGTase, the mutants had no cyclization activity but high hydrolysis and disproportionation activities. A possible mechanism for the enhanced substrate specificity was also analyzed through structure modeling of the mutant and wild-type CGTases. These results indicated that the -6 subsite played crucial roles in the substrate binding and catalytic reactions of CGTase and that the obtained CGTase mutants, especially Y167S/G179K/N193R/G180R, are promising starting points for further development through protein engineering.

Asunto(s)

Ácido Ascórbico/análogos & derivados , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Ingeniería Metabólica/métodos , Paenibacillus/enzimología , Polisacáridos/metabolismo , Ingeniería de Proteínas/métodos , Ácido Ascórbico/metabolismo , Sitios de Unión , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación Missense , Paenibacillus/genética , Conformación Proteica , Especificidad por Sustrato

RESUMEN

In this work, we integrated terminal truncation and N-terminal oligopeptide fusion as a novel protein engineering strategy to improve specific activity and catalytic efficiency of alkaline α-amylase (AmyK) from Alkalimonas amylolytica. First, the C terminus or N terminus of AmyK was partially truncated, yielding 12 truncated mutants, and then an oligopeptide (AEAEAKAKAEAEAKAK) was fused at the N terminus of the truncated AmyK, yielding another 12 truncation-fusion mutants. The specific activities of the truncation-fusion mutants AmyKΔC500-587::OP and AmyKΔC492-587::OP were 25.5- and 18.5-fold that of AmyK, respectively. The kcat/Km was increased from 1.0 × 10(5) liters · mol(-1) · s(-1) for AmyK to 30.6 × and 23.2 × 10(5) liters · mol(-1) · s(-1) for AmyKΔC500-587::OP and AmyKΔC492-587::OP, respectively. Comparative analysis of structure models indicated that the higher flexibility around the active site may be the main reason for the improved catalytic efficiency. The proposed terminal truncation and oligopeptide fusion strategy may be effective to engineer other enzymes to improve specific activity and catalytic efficiency.

Asunto(s)

Ingeniería de Proteínas/métodos , alfa-Amilasas/genética , alfa-Amilasas/metabolismo , Cinética , Modelos Moleculares , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Conformación Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia , Relación Estructura-Actividad

RESUMEN

In this work, we attempted to synthesize homoeriodictyol by transferring one methyl group of S-adenosyl-L-methionine (SAM) to eriodictyol using flavone 3'-O-methyltransferase ROMT-9, which was produced by recombinant Yarrowia lipolytica. Specifically, the ROMT-9 gene from rice was synthesized and cloned into the multi-copy integrative vector pINA1297, and was further expressed in Y. lipolytica with a growth phase-dependent constitutive promoter hp4d. The highest ROMT-9 activity reached 5.53 U/L after 4 days of culture in shake flask. The optimal pH and temperature of the purified ROMT-9 were 8.0 and 37 °C, respectively. The purified enzyme was stable up to 40 °C, and retained more than 80% of its maximal activity between pH 6.5 and 9.0. The recombinant ROMT-9 did not require Mg²âº for catalysis, while was completely inhibited in the presence of 5 mM Zn²âº, Cu²âº, Ba²âº, Al³âº, or Ni²âº. The purified ROMT-9 was used to synthesize homoeriodictyol, and the maximal transformation ratio reached 52.4% at 16 h under the following conditions: eriodictyol 0.2 g/L, ROMT-9 0.16 g/L, SAM 0.2 g/L, CH3OH 6% (v/v), temperature 37 °C, and pH 8.0. This work provides an alternative strategy for efficient synthesis of homoeriodictyol and compared to the traditional plant extraction or chemical synthesis, the biotransformation approach generates less environmental pollution and has a great potential for the sustainable production of homoeriodictyol.

Asunto(s)

Flavanonas/metabolismo , Flavonas/biosíntesis , Metiltransferasas/metabolismo , Yarrowia/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biotransformación , Clonación Molecular , Escherichia coli/genética , Expresión Génica , Vectores Genéticos , Metiltransferasas/genética , Oryza/enzimología , Oryza/genética , Proteínas Recombinantes/metabolismo , Yarrowia/genética

RESUMEN

Glucosamine (GlcN) and its acetylated derivative, N-acetylglucosamine (GlcNAc), are widely used in nutraceutical and pharmaceutical industries. Currently, GlcN and GlcNAc are mainly produced by hydrolysis from crab and shrimp shells, which can cause severe environmental pollution and carries the potential risk of allergic reactions. In this study, we attempted to achieve microbial production of GlcNAc by pathway engineering of Bacillus subtilis 168. Specifically, glmS (encoding GlcN-6-phosphate synthase) from B. subtilis 168 and GNA1 (encoding GlcNAc-6-phosphate N-acetyltransferase) from Saccharomyces cerevisiae S288C were firstly co-overexpressed in B. subtilis; the level of GlcNAc reached 240mg/L in shake flask culture. Next, nagP, encoding the GlcNAc-specific enzyme of phosphotransferase system, was deleted to block the importation of extracellular GlcNAC, thus improving GlcNAc production to 615mg/L in shake flask culture. Then, nagA (encoding GlcNAc-6-phosphate deacetylase), gamA (encoding GlcN-6-phosphate deaminase), and nagB (encoding GlcN-6-phosphate deaminase) were deleted to block the catabolism of intracellular GlcNAc, thereby further increasing the GlcNAc titer to 1.85g/L in shake flask culture. Finally, microbial production of GlcNAc by the engineered B. subtilis 168 was conducted in a 3-L fed-batch bioreactor, and the GlcNAc titer reached 5.19g/L, which was 2.8-fold of that in shake flask culture. This is the first report regarding the pathway engineering of B. subtilis for microbial production of GlcNAc, and provides a good starting point for further metabolic engineering to achieve the industrial production of GlcNAc by a generally regarded as safe strain.

Asunto(s)

Acetilglucosamina/biosíntesis , Bacillus subtilis/metabolismo , Ingeniería Metabólica , Acetilglucosamina/genética , Bacillus subtilis/genética , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Glucosamina 6-Fosfato N-Acetiltransferasa/biosíntesis , Glucosamina 6-Fosfato N-Acetiltransferasa/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/biosíntesis , Proteínas de Saccharomyces cerevisiae/genética

RESUMEN

Increasing concerns over limited petroleum resources and associated environmental problems are motivating the development of efficient cell factories to produce chemicals, fuels, and materials from renewable resources in an environmentally sustainable economical manner. Bacillus spp., the best characterized Gram-positive bacteria, possesses unique advantages as a host for producing microbial enzymes and industrially important biochemicals. With appropriate modifications to heterologous protein expression and metabolic engineering, Bacillus species are favorable industrial candidates for efficiently converting renewable resources to microbial enzymes, fine chemicals, bulk chemicals, and fuels. Here, we summarize the recent advances in developing Bacillus spp. as a cell factory. We review the available genetic tools, engineering strategies, genome sequence, genome-scale structure models, proteome, and secretion pathways, and we list successful examples of enzymes and industrially important biochemicals produced by Bacillus spp. Furthermore, we highlight the limitations and challenges in developing Bacillus spp. as a robust and efficient production host, and we discuss in the context of systems and synthetic biology the emerging opportunities and future research prospects in developing Bacillus spp. as a microbial cell factory.

Asunto(s)

Bacillus/genética , Bacillus/metabolismo , Proteínas Bacterianas/metabolismo , Microbiología Industrial , Biología Sintética , Bacillus/enzimología , Proteínas Bacterianas/genética , Biocombustibles

RESUMEN

In previous work, three evolved Propionibacterium acidipropionici mutants with higher tolerant capacity of propionic acid (PA) were obtained by genome shuffling. Here, we attempted to unravel the acid-tolerant mechanism of P. acidipropionici by comparing the physiological changes between P. acidipropionici and three mutants. The parameters used for comparison included intracellular pH (pHi), NAD⁺/NADH ratio, H⁺-ATPase activity, and the intracellular amino acids concentrations. It was indicated that the acid tolerance of P. acidipropionici was systematically regulated. Specifically, low pHi promoted the P. acidipropionici to biosynthesize more H⁺-ATPase to pump the protons out of the cells, and as a result, the NAD⁺/NADH ratio increased due to the decreased protons concentration. The increased arginine, aspartic acid, and glutamic acid concentrations helped to resist the acidic environment by consuming more H⁺ and generating more ATP and NH3. Based on what was analyzed above, 20 mM arginine and aspartic acid were added during the shaker culture of P. acidipropionici, and the maximal PA titer reached 14.38 g/L, which was increased by 39.9% compared with the control.

Asunto(s)

Propionatos/farmacología , Propionibacterium/metabolismo , Aminoácidos/metabolismo , Concentración de Iones de Hidrógeno , Mutación , NAD/metabolismo , Propionibacterium/efectos de los fármacos , Propionibacterium/genética , ATPasas de Translocación de Protón/metabolismo , Estrés Fisiológico

RESUMEN

In this work, the subsite-3 of cyclodextrin glycosyltransferase (CGTase) from Paenibacillus macerans was engineered to improve maltodextrin specificity for 2-O-d-glucopyranosyl-l-ascorbic acid (AA-2G) synthesis. Specifically, the site-saturation mutagenesis of tyrosine 89, asparagine 94, aspartic acid 196, and aspartic acid 372 in subsite-3 was separately performed, and three mutants Y89F (tyrosine→phenylalanine), N94P (asparagine→proline), and D196Y (aspartic acid→tyrosine) produced higher AA-2G titer than the wild-type and the other mutants. Previously, we found the mutant K47L (lysine→leucine) also had a higher maltodextrin specificity. Therefore, the four mutants K47L, Y89F, N94P, and D196Y were further used to construct the double, triple, and quadruple mutations. Among the 11 combinational mutants, the quadruple mutant K47L/Y89F/N94P/D196Y produced the highest AA-2G titer of 2.23g/L, which was increased by 85.8% compared to that produced by the wild-type CGTase. The reaction kinetics of all the mutants were modeled, and the pH and thermal stabilities of all the mutants were analyzed. The structure modeling indicated that the enhanced maltodextrin specificity may be related with the changes of hydrogen bonding interactions between the side chain of residue at the four positions (47, 89, 94, and 196) and the substrate sugars.

Asunto(s)

Ácido Ascórbico/análogos & derivados , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Mutagénesis Sitio-Dirigida/métodos , Paenibacillus/enzimología , Polisacáridos/metabolismo , Ingeniería de Proteínas , Sustitución de Aminoácidos/fisiología , Ácido Ascórbico/biosíntesis , Secuencia de Bases , Estabilidad de Enzimas/genética , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Paenibacillus/genética , Especificidad por Sustrato , Temperatura

RESUMEN

Microbial enzymes have been used in a large number of fields, such as chemical, agricultural and biopharmaceutical industries. The enzyme production rate and yield are the main factors to consider when choosing the appropriate expression system for the production of recombinant proteins. Recombinant enzymes have been expressed in bacteria (e.g., Escherichia coli, Bacillus and lactic acid bacteria), filamentous fungi (e.g., Aspergillus) and yeasts (e.g., Pichia pastoris). The favorable and very advantageous characteristics of these species have resulted in an increasing number of biotechnological applications. Bacterial hosts (e.g., E. coli) can be used to quickly and easily overexpress recombinant enzymes; however, bacterial systems cannot express very large proteins and proteins that require post-translational modifications. The main bacterial expression hosts, with the exception of lactic acid bacteria and filamentous fungi, can produce several toxins which are not compatible with the expression of recombinant enzymes in food and drugs. However, due to the multiplicity of the physiological impacts arising from high-level expression of genes encoding the enzymes and expression hosts, the goal of overproduction can hardly be achieved, and therefore, the yield of recombinant enzymes is limited. In this review, the recent strategies used for the high-level expression of microbial enzymes in the hosts mentioned above are summarized and the prospects are also discussed. We hope this review will contribute to the development of the enzyme-related research field.

Asunto(s)

Bacterias/metabolismo , Biotecnología/métodos , Proteínas Recombinantes/metabolismo , Bacterias/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Pichia/genética , Pichia/metabolismo

RESUMEN

Propionic acid (PA) is an important chemical building block and is widely applied for organic synthesis, food, feedstuff, and pharmaceuticals. To date, the strains that can efficiently produce PA have included Propionibacterium thoenii, P. freudenreichii, and P. acidipropionici. In this report, we show that P. jensenii ATCC 4868 is also able to produce PA in much higher yields than the previously reported strains. To further improve the production capacity, a P. jensenii-Escherichia coli shuttle vector was developed for the metabolic engineering of P. jensenii. Specifically, a 6.9-kb endogenous plasmid, pZGX01, was isolated from P. acidipropionici ATCC 4875 and sequenced. Since the sequencing analysis indicated that pZGX01 could encode 11 proteins, the transcriptional levels of the corresponding genes were also investigated. Then, a P. jensenii-Escherichia coli shuttle vector was constructed using the pZGX01 plasmid, the E. coli pUC18 plasmid, and a chloramphenicol resistance gene. Interestingly, not only could the developed shuttle vector be transformed into P. jensenii ATCC 4868 and 4870, but it also could be transformed into freudenreichii ATCC 6207 subspecies of P. freudenreichii. Finally, the glycerol dehydrogenase gene (gldA) from Klebsiella pneumoniae was expressed in P. jensenii ATCC 4868 with the constructed shuttle vector. In a 3-liter batch culture, the PA production by the engineered P. jensenii ATCC 4868 strain reached 28.23 ± 1.0 g/liter, which was 26.07% higher than that produced by the wild-type strain (22.06 ± 1.2 g/liter). This result indicated that the constructed vector can be used a useful tool for metabolic engineering of P. jensenii.

Asunto(s)

Escherichia coli/genética , Propionatos/metabolismo , Propionibacterium/genética , Clonación Molecular , Escherichia coli/metabolismo , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Ingeniería Metabólica , Datos de Secuencia Molecular , Plásmidos/genética , Plásmidos/metabolismo , Propionibacterium/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Análisis de Secuencia de Proteína

RESUMEN

In this study, we achieved the efficient synthesis of 2-O-d-glucopyranosyl-l-ascorbic acid (AA-2G) from soluble starch by fusing a carbohydrate-binding module (CBM) from Alkalimonas amylolytica α-amylase (CBMAmy) to cyclodextrin glycosyltransferase (CGTase) from Paenibacillus macerans. One fusion enzyme, CGT-CBMAmy, was constructed by fusing the CBMAmy to the C-terminal region of CGTase, and the other fusion enzyme, CGTΔE-CBMAmy, was obtained by replacing the E domain of CGTase with CBMAmy. The two fusion enzymes were then used to synthesize AA-2G from soluble starch as a cheap and easily soluble glycosyl donor. Under the optimal conditions, the AA-2G yields produced using CGTΔE-CBMAmy and CGT-CBMAmy were 2.01 g/liter and 3.03 g/liter, respectively, which were 3.94- and 5.94-fold of the yield from the wild-type CGTase (0.51 g/liter). The reaction kinetics of the two fusion enzymes were analyzed and modeled to confirm the enhanced specificity toward soluble starch. It was also found that, compared to the wild-type CGTase, the two fusion enzymes had relatively high hydrolysis and disproportionation activities, factors that favor AA-2G synthesis. Finally, it was speculated that the enhancement of soluble starch specificity may be related to the changes of substrate binding ability and the substrate binding sites between the CBM and the starch granule.

Asunto(s)

Ácido Ascórbico/análogos & derivados , Glucosiltransferasas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Almidón/metabolismo , Ácido Ascórbico/biosíntesis , Sitios de Unión , Escherichia coli/genética , Escherichia coli/metabolismo , Gammaproteobacteria/enzimología , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Glucosiltransferasas/genética , Hidrólisis , Simulación del Acoplamiento Molecular , Paenibacillus/enzimología , Proteínas Recombinantes de Fusión/genética , Solubilidad , alfa-Amilasas/genética , alfa-Amilasas/metabolismo

RESUMEN

In this study, we constructed and expressed six fusion proteins composed of oligopeptides attached to the N terminus of the alkaline α-amylase (AmyK) from Alkalimonas amylolytica. The oligopeptides had various effects on the functional and structural characteristics of AmyK. AmyK-p1, the fusion protein containing peptide 1 (AEAEAKAKAEAEAKAK), exhibited improved specific activity, catalytic efficiency, alkaline stability, thermal stability, and oxidative stability compared with AmyK. Compared with AmyK, the specific activity and catalytic constant (kcat) of AmyK-p1 were increased by 4.1-fold and 3.5-fold, respectively. The following properties were also improved in AmyK-p1 compared with AmyK: kcat/Km increased from 1.8 liter/(g·min) to 9.7 liter/(g·min), stable pH range was extended from 7.0 to 11.0 to 7.0 to 12.0, optimal temperature increased from 50°C to 55°C, and the half-life at 60°C increased by ∼2-fold. Moreover, AmyK-p1 showed improved resistance to oxidation and retained 54% of its activity after incubation with H2O2, compared with 20% activity retained by AmyK. Finally, AmyK-p1 was more compatible than AmyK with the commercial solid detergents tested. The mechanisms responsible for these changes were analyzed by comparing the three-dimensional (3-D) structural models of AmyK and AmyK-p1. The significantly enhanced catalytic efficiency and stability of AmyK-p1 suggests its potential as a detergent ingredient. In addition, the oligopeptide fusion strategy described here may be useful for improving the catalytic efficiency and stability of other industrial enzymes.

Asunto(s)

Gammaproteobacteria/enzimología , Oligopéptidos/metabolismo , alfa-Amilasas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Biocatálisis , Detergentes , Estabilidad de Enzimas , Gammaproteobacteria/genética , Expresión Génica , Semivida , Calor , Peróxido de Hidrógeno/farmacología , Concentración de Iones de Hidrógeno , Cinética , Modelos Estructurales , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Oligopéptidos/genética , Oxidación-Reducción , Conformación Proteica , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión , alfa-Amilasas/genética , alfa-Amilasas/aislamiento & purificación

RESUMEN

In this work, the site-saturation engineering of lysine 47 in cyclodextrin glycosyltransferase (CGTase) from Paenibacillus macerans was conducted to improve the specificity of CGTase towards maltodextrin, which can be used as a cheap and easily soluble glycosyl donor for the enzymatic synthesis of 2-O-D-glucopyranosyl-L-ascorbic acid (AA-2G) by CGTase. When using maltodextrin as glycosyl donor, four mutants K47F (lysine→ phenylalanine), K47L (lysine→ leucine), K47V (lysine→ valine) and K47W (lysine→ tryptophan) showed higher AA-2G yield as compared with that produced by the wild-type CGTase. The transformation conditions (temperature, pH and the mass ratio of L-ascorbic acid to maltodextrin) were optimized and the highest titer of AA-2G produced by the mutant K47L could reach 1.97 g/l, which was 64.2% higher than that (1.20 g/l) produced by the wild-type CGTase. The reaction kinetics analysis confirmed the enhanced maltodextrin specificity, and it was also found that compared with the wild-type CGTase, the four mutants had relatively lower cyclization activities and higher disproportionation activities, which was favorable for AA-2G synthesis. The mechanism responsible for the enhanced substrate specificity was further explored by structure modeling and it was indicated that the enhancement of maltodextrin specificity may be due to the short residue chain and the removal of hydrogen bonding interactions between the side chain of residue 47 and the sugar at -3 subsite. Here the obtained mutant CGTases, especially the K47L, has a great potential in the production of AA-2G with maltodextrin as a cheap and easily soluble substrate.

Asunto(s)

Ácido Ascórbico/análogos & derivados , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Lisina/genética , Mutagénesis Sitio-Dirigida , Paenibacillus/enzimología , Polisacáridos/metabolismo , Sustitución de Aminoácidos , Ácido Ascórbico/metabolismo , Estabilidad de Enzimas , Glucosiltransferasas/química , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Paenibacillus/genética , Conformación Proteica , Especificidad por Sustrato , Temperatura