[show abstract][hide abstract] ABSTRACT: Bioethanol production from various starchy materials has received much attention in recent years. alpha-Amylases are key enzymes in the bioconversion process of starchy biomass to biofuels, food or other products. The properties of thermostability, pH stability, and Ca-independency are important in the development of such fermentation process.
A novel Flavobacteriaceae Sinomicrobium alpha-amylase (FSA) was identified and characterized from genomic analysis of a novel Flavobacteriaceae species. It is closely related with archaeal alpha-amylases in the GH13_7 subfamily, but is evolutionary distant with other bacterial alpha-amylases. Based on the conserved sequence alignment and homology modeling, with minor variation, the Zn2+- and Ca2+-binding sites of FSA were predicated to be the same as those of the archaeal thermophilic alpha-amylases. The recombinant alpha-amylase was highly expressed and biochemically characterized. It showed optimum activity at pH 6.0, high enzyme stability at pH 6.0 to 11.0, but weak thermostability. A disulfide bond was introduced by site-directed mutagenesis in domain C and resulted in the apparent improvement of the enzyme activity at high temperature and broad pH range. Moreover, about 50% of the enzyme activity was detected under 100[degree sign]C condition, whereas no activity was observed for the wild type enzyme. Its thermostability was also enhanced to some extent, with the half-life time increasing from 25 to 55 minutes at 50[degree sign]C. In addition, after the introduction of the disulfide bond, the protein became a Ca-independent enzyme.
The improved stability of FSA suggested that the domain C contributes to the overall stability of the enzyme under extreme conditions. In addition, successfully directed modification and special evolutionary status of FSA imply its directional reconstruction potentials for bioethanol production, as well as for other industrial applications.
Biotechnology for Biofuels 01/2014; 7(1):18. · 5.55 Impact Factor
[show abstract][hide abstract] ABSTRACT: The production of biofuels by recombinant Escherichia coli is restricted by the toxicity of the products. 2,3-Butanediol (2,3-BD), a platform and fuel bio-chemical with low toxicity to microbes, could be a promising alternative for biofuel production. However, the yield and productivity of 2,3-BD produced by recombinant E. coli strains are not sufficient for industrial scale fermentation. In this work, the production of 2,3-BD by recombinant E. coli strains was optimized by applying a systematic approach. 2,3-BD biosynthesis gene clusters were cloned from several native 2,3-BD producers, including Bacillus subtilis, Bacillus licheniformis, Klebsiella pneumoniae, Serratia marcescens, and Enterobacter cloacae, inserted into the expression vector pET28a, and compared for 2,3-BD synthesis. The recombinant strain E. coli BL21/pETPT7-EcABC, carrying the 2,3-BD pathway gene cluster from Enterobacter cloacae, showed the best ability to synthesize 2,3-BD. Thereafter, expression of the most efficient gene cluster was optimized by using different promoters, including PT7, Ptac, Pc, and Pabc. E. coli BL21/pET-RABC with Pabc as promoter was superior in 2,3-BD synthesis. On the basis of the results of biomass and extracellular metabolite profiling analyses, fermentation conditions, including pH, agitation speed, and aeration rate, were optimized for the efficient production of 2,3-BD. After fed-batch fermentation under the optimized conditions, 73.8 g/L of 2,3-BD was produced by using E. coli BL21/pET-RABC within 62 h. The values of both yield and productivity of 2,3-BD obtained with the optimized biological system were the highest ever achieved with an engineered E. coli strain. In addition to the 2,3-BD production, the systematic approach might also be used in the production of other important chemicals through recombinant E. coli strains.
[show abstract][hide abstract] ABSTRACT: In this study, a food-grade cell surface display host/vector system for Lactobacillus casei was constructed. The food-grade host L. casei Q-5 was a lactose-deficient derivative of L. casei ATCC 334 obtained by plasmid elimination. The food-grade cell surface display vector was constructed based on safe DNA elements from lactic acid bacteria containing the following: pSH71 replicon from Lactococcus lactis, lactose metabolism genes from L. casei ATCC 334 as complementation markers, and surface layer protein gene from Lactobacillus acidophilus ATCC 4356 for cell surface display. The feasibility of the new host/vector system was verified by the expression of green fluorescent protein (GFP) on L. casei. Laser scanning confocal microscopy and immunofluorescence analysis using anti-GFP antibody confirmed that GFP was anchored on the surface of the recombinant cells. The stability of recombinant L. casei cells in artificial gastrointestinal conditions was verified, which is beneficial for oral vaccination applications. These results indicate that the food-grade host/vector system can be an excellent antigen delivery vehicle in oral vaccine construction.
Microbiological Research 01/2014; · 1.99 Impact Factor
[show abstract][hide abstract] ABSTRACT: 2,3-Butanediol is a platform and fuel biochemical that can be efficiently produced from biomass. However, a value-added process for this chemical has not yet been developed. To expand the utilization of 2,3-butanediol produced from biomass, an improved derivative process of 2,3-butanediol is desirable.
In this study, a Gluconobacter oxydans strain DSM 2003 was found to have the ability to transform 2,3-butanediol into acetoin, a high value feedstock that can be widely used in dairy and cosmetic products, and chemical synthesis. All three stereoisomers, meso-2,3-butanediol, (2R,3R)-2,3-butanediol, and (2S,3S)-2,3-butanediol, could be transformed into acetoin by the strain. After optimization of the bioconversion conditions, the optimum growth temperature for acetoin production by strain DSM 2003 was found to be 30[degree sign]C and the medium pH was 6.0. With an initial 2,3-butanediol concentration of 40 g/L, acetoin at a high concentration of 89.2 g/L was obtained from 2,3-butanediol by fed-batch bioconversion with a high productivity (1.24 g/L . h) and high yield (0.912 mol/mol).
G. oxydans DSM 2003 is the first strain that can be used in the direct production of acetoin from 2,3-butanediol. The product concentration and yield of the novel process are both new records for acetoin production. The results demonstrate that the method developed in this study could provide a promising process for efficient acetoin production and industrially produced 2,3-butanediol utilization.
Biotechnology for Biofuels 10/2013; 6(1):155. · 5.55 Impact Factor
[show abstract][hide abstract] ABSTRACT: Fermentative production of lactic acid, an important bio-based chemicals, has made considerable progress. In addition to the food industry and production of polylactic acid, lactic acid also can be used as an important platform chemical for the production of acrylic acid, pyruvic acid, 1,2-propanediol, and lactic acid esters. This article summarizes the recent progress in biocatalytic production of lactic acid derivatives by dehydration, dehydrogenation, reduction, and esterification. Trends in the biotransformation of lactic acid are also discussed.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 10/2013; 29(10):1411-20.
[show abstract][hide abstract] ABSTRACT: (2S,3S)-2,3-Butanediol ((2S,3S)-2,3-BD) is a potentially valuable liquid fuel and an excellent building block in asymmetric synthesis. In this study, cofactor engineering was applied to improve the efficiency of (2S,3S)-2,3-BD production and simplify the product purification. Two NADH regeneration enzymes, glucose dehydrogenase and formate dehydrogenase (FDH), were introduced into Escherichia coli with 2,3-BD dehydrogenase, respectively. Introduction of FDH resulted in higher (2S,3S)-2,3-BD concentration, productivity and yield from diacetyl, and large increase in the intracellular NADH concentration. In fed-batch bioconversion, the final titer, productivity and yield of (2S,3S)-2,3-BD on diacetyl reached 31.7 g/L, 2.3 g/(L·h) and 89.8%, the highest level of (2S,3S)-2,3-BD production thus far. Moreover, cosubstrate formate was almost totally converted to carbon dioxide and no organic acids were produced. The biocatalytic process presented should be a promising route for biotechnological production of NADH-dependent microbial metabolites.
[show abstract][hide abstract] ABSTRACT: 2,3-Butanediol (2,3-BD), a platform and fuel bio-chemical, can be efficiently produced by Klebsiella pneumonia, K. oxytoca, and Serratia marcescens. However, these strains are opportunistic pathogens and not favorable for industrial application. Although some generally regarded as safe (GRAS) microorganisms have been isolated in recent years, there is still a demand for safe 2,3-BD producing strains with high productivity and yield under thermophilic fermentation.
Bacillus licheniformis strain 10-1-A was newly isolated for 2,3-BD production. The optimum temperature and medium pH were 50[degree sign]C and pH 7.0 for 2,3-BD production by strain 10-1-A. The medium composition was optimized through Plackett--Burman design and response surface methodology techniques. With a two-stage agitation speed control strategy, 115.7 g/L of 2,3-BD was obtained from glucose by fed-batch fermentation in a 5-L bioreactor with a high productivity (2.4 g/L.h) and yield (94% of its theoretical value). The 2,3-BD produced by strain 10-1-A comprises (2R,3R)-2,3-BD and meso-2,3-BD with a ratio of nearly 1:1. The bdh and gdh genes encoding meso-2,3-butanediol dehydrogenase (meso-BDH) and glycerol dehydrogenase (GDH) of strain 10-1-A were expressed in Escherichia coli and the proteins were purified. meso-2,3-BD and (2R,3R)-2,3-BD were transformed from racemic acetoin by meso-BDH and GDH with NADH, respectively.
Compared with the reported GRAS 2,3-BD producers, B. licheniformis 10-1-A could thermophilically produce 2,3-BD with a high concentration, productivity and yield. Thus, the newly isolated GRAS strain 10-1-A might be a promising strain for industrial production of 2,3-BD. Two key enzymes for meso-2,3-BD and (2R,3R)-2,3-BD production were purified and further studied, and this might be helpful to understand the mechanism for 2,3-BD stereoisomers forming in B. licheniformis.
Biotechnology for Biofuels 08/2013; 6(1):123. · 5.55 Impact Factor
[show abstract][hide abstract] ABSTRACT: Escherichia coli NusA, an essential component of the RNA polymerase elongation complex, is involved in transcriptional elongation, termination, anti-termination, cold shock and stress-induced mutagenesis. In this study, we demonstrated that NusA can self-assemble into oligomers under heat shock conditions and that this property is largely determined by the C-terminal domain. In parallel with the self-assembly process, NusA also acquires chaperone activity. Furthermore, NusA overexpression results in the enhanced heat shock resistance of host cells, which may be due to the chaperone activity of NusA. Our results suggest that E. coli NusA can act as a protector to prevent protein aggregation under heat stress conditions in vitro and in the NusA-overexpressing strain. We propose a new hypothesis that NusA could serve as a molecular chaperone in addition to its functions as a transcription factor. However, it remains to be further investigated whether NusA has the same function under normal physiological conditions.
[show abstract][hide abstract] ABSTRACT: Production of (3S)-acetoin ((3S)-AC), an important platform chemical, is desirable but difficult to perform. An NADPH-dependent carbonyl reductase (Gox0644) from Gluconobacter oxydans DSM 2003 was confirmed to have a good ability to reduce diacetyl (DA) to produce (3S)-AC. In this work, the NADPH-dependent carbonyl reductase was expressed and purified. Glucose dehydrogenase from Bacillus subtilis 168 was coupled with the NADPH-dependent carbonyl reductase to produce (3S)-AC from DA. Under the optimal conditions, 12.2gl(-1) (3S)-AC was produced from 14.3gl(-1) DA in 75min. Because DA can be biotechnological produced, the two-enzymes coupling system might be a promising alternative for the (3S)-AC production.
[show abstract][hide abstract] ABSTRACT: In this work, bio-oxidation of glycerate was introduced for the green production of hydroxypyruvate. Whole cells of Pseudomonas sp. XP-LM were confirmed to have a good ability to produce hydroxypyruvate from glycerate. Under the optimal conditions, Hydroxypyruvate of 98.6mM was produced from glycerate of 200mM. Glycerate is now potentially producible from glycerol, a by-product during biodiesel fuel production, through a biotechnological process. Thus, the bioconversion system introduced in this work provided not only a green hydroxypyruvate production process but also a potential pathway of surplus glycerol utilization.
[show abstract][hide abstract] ABSTRACT: Constitutive vectors are useful tools for genetic engineering. Two constitutive vectors, with high expression and broad host range, were developed and used in a range of Pseudomonas hosts. The vectors showed superior characteristics compared to the inducible vectors as well as the potential to be well-improved genetic tools for biocatalysis.
Applied and environmental microbiology 02/2013; · 3.69 Impact Factor
[show abstract][hide abstract] ABSTRACT: The enzyme 6-phospho-β-glucosidase is an important member in the glycoside hydrolase family 1 (GH1). However, its catalytic mechanisms, especially the key residues determining substrate specificity and affinity are poorly understood. A metagenome-derived gene sequence, encoding a novel 6-phospho-β-glucosidase designated as Pbgl25-217, was isolated and characterized. Optimal conditions for enzymatic activity were 37°C and pH 7; Ca(2+), Mg(2+), and Mn(2+) stabilized the activity of Pbgl25-217, whereas Ni(2+), Fe(2+), Zn(2+), Cu(2+), and Fe(3+) inhibited its activity. The K(m) and V(max) values of Pbgl25-217 were 4.8 mM and 1987.0 U⋅mg(-1), respectively. Seven conserved residues were recognized by multiple alignments, and were tested by site-directed mutagenesis for their functions in substrate recognition and catalytic reaction. Results suggests that residues of S427, Lys435, and Tyr437 act as the "gatekeepers" in a phosphate-binding loop, and play important roles in phosphate recognition. This functional identification may provide insights into the specificity of 6-phospho-β-glycosidases in GH1 and useful for designing further directed evolution.
Applied and environmental microbiology 01/2013; · 3.69 Impact Factor
[show abstract][hide abstract] ABSTRACT: Clostridium butyricum strains have been considered promising producers of biofuels and biochemicals, such as hydrogen, butanol, butyric acid, and 1,3-propanediol. Here, we present a 4.59-Mb assembly of the genome sequence of DSM 10702 (VPI 3266), a type strain of C. butyricum.
[show abstract][hide abstract] ABSTRACT: An NAD-dependent d-lactate dehydrogenase (d-nLDH) of Lactobacillus bulgaricus ATCC 11842 was rationally re-designed for asymmetric reduction of a homologous series of α-keto carboxylic acids such as phenylpyruvic acid (PPA), α-ketobutyric acid, α-ketovaleric acid, β-hydroxypyruvate. Compared with wild-type d-nLDH, the Y52L mutant d-nLDH showed elevated activities toward unnatural substrates especially with large substitutes at C-3. By the biocatalysis combined with a formate dehydrogenase for in situ generation of NADH, the corresponding (R)-α-hydroxy carboxylic acids could be produced at high yields and highly optical purities. Taking the production of chiral (R)-phenyllactic acid (PLA) from PPA for example, 50 mM PPA was completely reduced to (R)-PLA in 90 min with a high yield of 99.0% and a highly optical purity (>99.9% e.e.) by the coupling system. The results presented in this work suggest a promising alternative for the production of chiral α-hydroxy carboxylic acids.
[show abstract][hide abstract] ABSTRACT: BACKGROUND: NAD-independent l-lactate dehydrogenase (l-iLDH) from Pseudomonas stutzeri SDM can potentially be used for the kinetic resolution of small aliphatic 2-hydroxycarboxylic acids. However, this enzyme showed rather low activity towards aromatic 2-hydroxycarboxylic acids. RESULTS: Val-108 of l-iLDH was changed to Ala by rationally site-directed mutagenesis. The l-iLDH mutant exhibited much higher activity than wide-type l-iLDH towards l-mandelate, an aromatic 2-hydroxycarboxylic acid. Using the engineered Escherichia coli expressing the mutant l-iLDH as a biocatalyst, 40 g.L-1 of dl-mandelic acid was converted to 20.1 g.L-1 of d-mandelic acid (enantiomeric purity higher than 99.5%) and 19.3 g.L-1 of benzoylformic acid. CONCLUSIONS: A new biocatalyst with high catalytic efficiency toward an unnatural substrate was constructed by rationally re-design mutagenesis. Two building block intermediates (optically pure d-mandelic acid and benzoylformic acid) were efficiently produced by the one-pot biotransformation system.
[show abstract][hide abstract] ABSTRACT: Pulp mill wastewater generated from wheat straw is characterized as high alkalinity and very high COD pollution load. A naturally developed microbial community in a pulp mill wastewater storage pool that had been disused were investigated in this study. Owing to natural evaporation and a huge amount of lignocellulose's deposition, the wastewater sediment contains high concentrations of organic matters and sodium ions, but low concentrations of chloride and carbonate. The microbiota inhabiting especially anaerobic community, including methanogenic arhcaea and cellulolytic species, was studied. All archaeal sequences fall into 2 clusters of family Halobacteriaceae and methanogenic archaeon in the phylum Euryarchaeota. In the methanogenic community, phylogenetic analysis of methyl coenzyme M reductase A (mcrA) genes targeted to novel species in genus Methanoculleus or novel genus of order Methanomicrobiales. The predominance of Methanomicrobiales suggests that methanogenesis in this system might be driven by the hydrogenotrophic pathway. As the important primary fermenter for methane production, the cellulolytic community of enzyme GHF48 was found to be dominated by narrower breadth of novel clostridial cellulase genes. Novel anoxic functional members in such extreme sediment provide the possibility of enhancing the efficiency of anoxic treatment of saline and alkaline wastewaters, as well as benefiting to the biomass transformation and biofuel production processes.
[show abstract][hide abstract] ABSTRACT: Xanthomonas campestris JX, a soil bacterium, is an industrially productive strain for xanthan gum. Here we present a 5.0-Mb assembly of its genome sequence. We have annotated 12 coding sequences (CDSs) responsible for xanthan gum biosynthesis, 346 CDSs encoding carbohydrate metabolism, and 69 CDSs related to virulence, defense, and plant disease.
Journal of bacteriology 09/2012; 194(17):4755-6. · 3.94 Impact Factor
[show abstract][hide abstract] ABSTRACT: Pseudomonas aeruginosa XMG, isolated from soil, utilizes lactate. Here we present a 6.45-Mb assembly of its genome sequence. Besides the lactate utilization mechanism of the strain, the genome sequence may also provide other useful information related to P. aeruginosa, such as identifying genes involved in virulence, drug resistance, and aromatic catabolism.
Journal of bacteriology 09/2012; 194(17):4751-2. · 3.94 Impact Factor
[show abstract][hide abstract] ABSTRACT: Klebsiella pneumoniae LZ is a bacterium isolated from soil which can produce 1,3-propanediol from glycerol. Here we present a 5,431,750-bp assembly of its genome sequence. We annotated 9 coding sequences (CDSs) responsible for glycerol fermentation to 1,3-propanediol, 19 CDSs encoding glycerol utilization, and 134 CDSs related to its virulence and defense.
Journal of bacteriology 08/2012; 194(16):4457-8. · 3.94 Impact Factor