Engineering of a Stable Whole-Cell Biocatalyst Capable of (S)-Styrene Oxide Formation for Continuous Two-Liquid-Phase Applications

Recombinant strains of Pseudomonas putida KT2440 carrying genetic expression cassettes with xylene oxygenase- and styrene monooxygenase-encoding genes on their chromosomes could be induced in shaking-flask experiments to specific activities that rivaled those of multicopy-plasmid-based Escherichia coli recombinants. Such strains maintained the introduced styrene oxidation activity in continuous two-liquid-phase cultures for at least 100 generations, although at a lower level than in the shaking-flask experiments. The data suggest that placement of target genes on the chromosome might be a suitable route for the construction of segregationally stable and highly active whole-cell biocatalysts.     

A Novel Whole-Cell Biocatalyst with NAD+ Regeneration for Production of Chiral Chemicals

Background

The high costs of pyridine nucleotide cofactors have limited the applications of NAD(P)-dependent oxidoreductases on an industrial scale. Although NAD(P)H regeneration systems have been widely studied, NAD(P)⁺ regeneration, which is required in reactions where the oxidized form of the cofactor is used, has been less well explored, particularly in whole-cell biocatalytic processes.

Methodology/Principal Findings

Simultaneous overexpression of an NAD⁺ dependent enzyme and an NAD⁺ regenerating enzyme (H₂O producing NADH oxidase from Lactobacillus brevis) in a whole-cell biocatalyst was studied for application in the NAD⁺-dependent oxidation system. The whole-cell biocatalyst with (2R,3R)-2,3-butanediol dehydrogenase as the catalyzing enzyme was used to produce (3R)-acetoin, (3S)-acetoin and (2S,3S)-2,3-butanediol.

Conclusions/Significance

A recombinant strain, in which an NAD⁺ regeneration enzyme was coexpressed, displayed significantly higher biocatalytic efficiency in terms of the production of chiral acetoin and (2S,3S)-2,3-butanediol. The application of this coexpression system to the production of other chiral chemicals could be extended by using different NAD(P)-dependent dehydrogenases that require NAD(P)⁺ for catalysis.     

Development of an Autofluorescent Whole-Cell Biocatalyst by Displaying Dual Functional Moieties on Escherichia coli Cell Surfaces and Construction of a Coculture with Organophosphate-Mineralizing Activity

Surface display of the active proteins on living cells has enormous potential in the degradation of numerous toxic compounds. Here, we report the codisplay of organophosphorus hydrolase (OPH) and enhanced green fluorescent protein (GFP) on the cell surface of Escherichia coli by use of the truncated ice nucleation protein (INPNC) and Lpp-OmpA fusion systems. The surface localization of both INPNC-OPH and Lpp-OmpA-GFP was demonstrated by Western blot analysis, immunofluorescence microscopy, and a protease accessibility experiment. Anchorage of GFP and OPH on the outer membrane neither inhibits cell growth nor affects cell viability, as shown by growth kinetics of cells and stability of resting cultures. The engineered E. coli can be applied in the form of a whole-cell biocatalyst and can be tracked by fluorescence during bioremediation. This strategy of codisplay should open a new dimension for the display of multiple functional moieties on the surface of a bacterial cell. Furthermore, a coculture comprised of the engineered E. coli and a natural p-nitrophenol (PNP) degrader, Ochrobactrum sp. strain LL-1, was assembled for complete mineralization of organophosphates (OPs) with a PNP substitution. The coculture degraded OPs as well as PNP rapidly. Therefore, the coculture with autofluorescent and mineralizing activities can potentially be applied for bioremediation of OP-contaminated sites.     

Multicolor Whole-Cell Bacterial Sensing Using a Synchronous Fluorescence Spectroscopy-Based Approach

The wide collection of currently available fluorescent proteins (FPs) offers new possibilities for multicolor reporter gene-based studies of bacterial functions. However, the simultaneous use of multiple FPs is often limited by the bleed-through of their emission spectra. Here we introduce an original approach for detection and separation of multiple overlapping fluorescent signals from mixtures of bioreporters strains. The proposed method relies on the coupling of synchronous fluorescent spectroscopy (SFS) with blind spectral decomposition achieved by the Canonical Polyadic (CP) decomposition (also known as Candecomp/Parafac) of three-dimensional data arrays. Due to the substantial narrowing of FP emission spectra and sensitive detection of multiple FPs in a one-step scan, SFS reduced spectral overlap and improved the selectivity of the CP unmixing procedure. When tested on mixtures of labeled E. coli strains, the SFS/CP approach could easily extract the contribution of at least four overlapping FPs. Furthermore, it allowed to simultaneously monitor the expression of three iron responsive genes and pyoverdine production in P. aeruginosa. Implemented in a convenient microplate format, this multiplex fluorescent reporter method provides a useful tool to study complex processes with different variables in bacterial systems.     

Continuous Asymmetric Reduction Of 4-Oxoisophorone By Thermophilic Bacteria Using A Hollow Fiber Reactor

Continuous asymmetric reduction of 4-oxoisophorone by the thermophilic bacterium Thermomonospora curvata JTS321 was examined using three reactor systems: packed bed, fluidized bed and hollow fiber. T. curvata was immobilized in polyacrylamide-hydrazide gels when used in the packed and fluidized bed reactors. Of the three reactor systems, the highest productivity (964 mg.1^-1.h^-1) was observed in the fluidized bed reactor. However, many cells grew outside of the gel matrix, causing product contamination. The productivity of the hollow fiber reactor was 504 mg.1^-1.h^-1; the problem of cell contamination of the product was avoided, as the molecular cut-off of the hollow fibers (400 000) was of an appropriate size to prevent cell leakage to the product stream. We therefore consider that the hollow fiber reactor is most suitable for continuous microbial conversions.     

Continuous Asymmetric Reduction Of 4-Oxoisophorone By Thermophilic Bacteria Using A Hollow Fiber Reactor

Continuous asymmetric reduction of 4-oxoisophorone by the thermophilic bacterium Thermomonospora curvata JTS321 was examined using three reactor systems: packed bed, fluidized bed and hollow fiber. T. curvata was immobilized in polyacrylamide-hydrazide gels when used in the packed and fluidized bed reactors. Of the three reactor systems, the highest productivity (964 mg.1^-1.h^-1) was observed in the fluidized bed reactor. However, many cells grew outside of the gel matrix, causing product contamination. The productivity of the hollow fiber reactor was 504 mg.1^-1.h^-1; the problem of cell contamination of the product was avoided, as the molecular cut-off of the hollow fibers (400 000) was of an appropriate size to prevent cell leakage to the product stream. We therefore consider that the hollow fiber reactor is most suitable for continuous microbial conversions.     

Substrate-Specific Development of Thermophilic Bacterial Consortia by Using Chemically Pretreated Switchgrass

Microbial communities that deconstruct plant biomass have broad relevance in biofuel production and global carbon cycling. Biomass pretreatments reduce plant biomass recalcitrance for increased efficiency of enzymatic hydrolysis. We exploited these chemical pretreatments to study how thermophilic bacterial consortia adapt to deconstruct switchgrass (SG) biomass of various compositions. Microbial communities were adapted to untreated, ammonium fiber expansion (AFEX)-pretreated, and ionic-liquid (IL)-pretreated SG under aerobic, thermophilic conditions using green waste compost as the inoculum to study biomass deconstruction by microbial consortia. After microbial cultivation, gravimetric analysis of the residual biomass demonstrated that both AFEX and IL pretreatment enhanced the deconstruction of the SG biomass approximately 2-fold. Two-dimensional nuclear magnetic resonance (2D-NMR) experiments and acetyl bromide-reactive-lignin analysis indicated that polysaccharide hydrolysis was the dominant process occurring during microbial biomass deconstruction, and lignin remaining in the residual biomass was largely unmodified. Small-subunit (SSU) rRNA gene amplicon libraries revealed that although the dominant taxa across these chemical pretreatments were consistently represented by members of the Firmicutes, the Bacteroidetes, and Deinococcus-Thermus, the abundance of selected operational taxonomic units (OTUs) varied, suggesting adaptations to the different substrates. Combining the observations of differences in the community structure and the chemical and physical structure of the biomass, we hypothesize specific roles for individual community members in biomass deconstruction.     

Development of Molecular Identification Method for Genus Alexandrium (Dinophyceae) Using Whole-Cell FISH

We have developed a method to identify species in the genus Alexandrium using whole-cell fluorescent in situ hybridization with FITC-labeled oligonucleotide probes that target large subunit ribosomal rRNA molecules. The probes were designed based on the sequence of the rDNA D1-D2 region of Alexandrium species. DNA probes specific for toxic A. tamarense and A. catenella and nontoxic A. affine, A. fraterculus, A. insuetum, and A. pseudogonyaulax, respectively, were applied to vegetative cells of all above Alexandrium species to test the sensitivity of the probes. Each DNA probe hybridized specifically with vegetative cells of the corresponding Alexandrium species and showed no cross-reactivity to noncorresponding Alexandrium species. In addition, no cross-reactivity of the probes was observed in experiments using concentrated natural seawater samples. The TAMAD2 probe, which is highly specific to A. tamarense, a common toxic species in Korean coastal waters, provides a simple and reliable molecular tool for identification of toxic Alexandrium species.     

Enhanced Reactivity of Rhizopus oryzae Lipase Displayed on Yeast Cell Surfaces in Organic Solvents: Potential as a Whole-Cell Biocatalyst in Organic Solvents

Immobilization of enzymes on some solid supports has been used to stabilize enzymes in organic solvents. In this study, we evaluated applications of genetically immobilized Rhizopus oryzae lipase displayed on the cell surface of Saccharomyces cerevisiae in organic solvents and measured the catalytic activity of the displayed enzyme as a fusion protein with α-agglutinin. Compared to the activity of a commercial preparation of this lipase, the activity of the new preparation was 4.4 × 10⁴-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate and 3.8 × 10⁴-fold higher in an esterification reaction with palmitic acid and n-pentanol (0.2% H₂O). Increased enzyme activity may occur because the lipase displayed on the yeast cell surface is stabilized by the cell wall. We used a combination of error-prone PCR and cell surface display to increase lipase activity. Of 7,000 colonies in a library of mutated lipases, 13 formed a clear halo on plates containing 0.2% methyl palmitate. In organic solvents, the catalytic activity of 5/13 mutants was three- to sixfold higher than that of the original construct. Thus, yeast cells displaying the lipase can be used in organic solvents, and the lipase activity may be increased by a combination of protein engineering and display techniques. Thus, this immobilized lipase, which is more easily prepared and has higher activity than commercially available free and immobilized lipases, may be a practical alternative for the production of esters derived from fatty acids.     

Whole-Cell Protein Identification Using the Concept of Unique Peptides

A concept of unique peptides(CUP)was proposed and implemented to identify whole-cell proteins from tandem mass spectrometry(MS/MS)ion spectra.A unique peptide is defined as a peptide,irrespective of its length,that exists only in one protein of a proteome of interest,despite the fact that this peptide may appear more than once in the same protein.Integrating CUP,a two-step whole-cell protein identification strategy was developed to further increase the confidence of identified proteins.A dataset containing 40,243 MS/MS ion spectra of Saccharomyces cerevisiae and protein identification tools including Mascot and SEQUEST were used to illustrate the proposed concept and strategy.Without implementing CUP,the proteins identified by SEQUEST are 2.26 fold of those identified by Mascot.When CUP was applied,the proteins bearing unique peptides identified by SEQUEST are3.89 fold of those identified by Mascot.By cross-comparing two sets of identified proteins,only 89 common proteins derived from CUP were found.The key discrepancy between identified proteins was resulted from the filtering criteria employed by each protein identification tool.According to the origin of peptides classified by CUP and the commonality of proteins recognized by protein identification tools,all identified proteins were cross-compared,resulting in four groups of proteins possessing different levels of assigned confidence.     

Efficient Whole-Cell Biocatalyst for Acetoin Production with NAD+ Regeneration System through Homologous Co-Expression of 2,3-Butanediol Dehydrogenase and NADH Oxidase in Engineered Bacillus subtilis

Acetoin (3-hydroxy-2-butanone), an extensively-used food spice and bio-based platform chemical, is usually produced by chemical synthesis methods. With increasingly requirement of food security and environmental protection, bio-fermentation of acetoin by microorganisms has a great promising market. However, through metabolic engineering strategies, the mixed acid-butanediol fermentation metabolizes a certain portion of substrate to the by-products of organic acids such as lactic acid and acetic acid, which causes energy cost and increases the difficulty of product purification in downstream processes. In this work, due to the high efficiency of enzymatic reaction and excellent selectivity, a strategy for efficiently converting 2,3-butandiol to acetoin using whole-cell biocatalyst by engineered Bacillus subtilis is proposed. In this process, NAD⁺ plays a significant role on 2,3-butanediol and acetoin distribution, so the NADH oxidase and 2,3-butanediol dehydrogenase both from B. subtilis are co-expressed in B. subtilis 168 to construct an NAD⁺ regeneration system, which forces dramatic decrease of the intracellular NADH concentration (1.6 fold) and NADH/NAD⁺ ratio (2.2 fold). By optimization of the enzymatic reaction and applying repeated batch conversion, the whole-cell biocatalyst efficiently produced 91.8 g/L acetoin with a productivity of 2.30 g/(L·h), which was the highest record ever reported by biocatalysis. This work indicated that manipulation of the intracellular cofactor levels was more effective than the strategy of enhancing enzyme activity, and the bioprocess for NAD⁺ regeneration may also be a useful way for improving the productivity of NAD⁺-dependent chemistry-based products.     

Distribution of Microorganisms in Deep-Sea Hydrothermal Vent Chimneys Investigated by Whole-Cell Hybridization and Enrichment Culture of Thermophilic Subpopulations

The microbial community structure of hydrothermal vent chimneys was evaluated by the combined use of enrichment cultures and whole-cell hybridizations with fluorescently labeled 16S rRNA-based oligonucleotide probes. Chimneys were collected during the Microsmoke cruise on the Mid-Atlantic Ridge and were subsampled on board and stored under reduced conditions or fixed. For estimation of culturable thermophiles, selective media were inoculated by dilution series of the samples and incubated at 65, 80, and 95(deg)C. To analyze the microbial diversity of the samples, cells were extracted from the fixed chimney structure samples and hybridized with domain- and kingdom-specific probes. Quantification of the extracted cells was assessed by whole-cell hybridization on membrane filters. By both methods, the largest amounts of microorganisms were found in the upper and outer parts of the chimneys, although even the inner parts contained culturable and detectable amounts of cells. Different morphotypes of thermophilic and hyperthermophilic microorganisms were enriched and detected in samples. Our data clearly indicate that the morphological diversity observed by using whole-cell hybridization is much larger than that assessed by use of culture-based enrichments. This new approach, including culture-independent and -dependent methods to study hydrothermal vent chimneys, showed an uneven distribution of a diverse microbial community. Application of lower-level specific probes for known families and genera within each domain by our approach will be useful to reveal the real extent and nature of the chimney microbial diversity and to support cultivation attempts.     

Comparative Biochemical and Structural Properties of an Industrially Important Biocatalyst Cellobiohydrolase Cel7A from Thermophilic Aspergillus fumigatus

Applied Biochemistry and Microbiology - Cellobiohydrolase GH7 (Cel7A) is a crucial component of an efficient cellulase system. High cellulase activity was identified in the secretome of a locally...     

生物催化剂与酶概念的发展

回顾了酶的研究历史 .综述了各种新兴“酶” ,特别是酶性RNA和酶性DNA的研究进展 .对各种“酶”与生物催化剂的概念的归属问题进行了探讨 .     

宏基因组学在新型生物催化剂开发中的研究进展

宏基因组学是基因工程发展的新方向,它为寻找和发现新的功能基因及生物催化剂提供了新的研究策略。着重论述了宏基因组学的研究方法,包括DNA的提取、文库的构建以及筛选策略的选择。同时介绍了近年来宏基因组学应用于新型生物催化剂开发中所取得的一些成果。     

Isolation and Screening of Thermophilic Bacilli from Compost for Electrotransformation and Fermentation: Characterization of Bacillus smithii ET 138 as a New Biocatalyst

Thermophilic bacteria are regarded as attractive production organisms for cost-efficient conversion of renewable resources to green chemicals, but their genetic accessibility is a major bottleneck in developing them into versatile platform organisms. In this study, we aimed to isolate thermophilic, facultatively anaerobic bacilli that are genetically accessible and have potential as platform organisms. From compost, we isolated 267 strains that produced acids from C₅ and C₆ sugars at temperatures of 55°C or 65°C. Subsequently, 44 strains that showed the highest production of acids were screened for genetic accessibility by electroporation. Two Geobacillus thermodenitrificans isolates and one Bacillus smithii isolate were found to be transformable with plasmid pNW33n. Of these, B. smithii ET 138 was the best-performing strain in laboratory-scale fermentations and was capable of producing organic acids from glucose as well as from xylose. It is an acidotolerant strain able to produce organic acids until a lower limit of approximately pH 4.5. As genetic accessibility of B. smithii had not been described previously, six other B. smithii strains from the DSMZ culture collection were tested for electroporation efficiencies, and we found the type strain DSM 4216^T and strain DSM 460 to be transformable. The transformation protocol for B. smithii isolate ET 138 was optimized to obtain approximately 5 × 10³ colonies per μg plasmid pNW33n. Genetic accessibility combined with robust acid production capacities on C₅ and C₆ sugars at a relatively broad pH range make B. smithii ET 138 an attractive biocatalyst for the production of lactic acid and potentially other green chemicals.     

Whole-Cell Analysis of Low-Density Lipoprotein Uptake by Macrophages Using STEM Tomography

Nanoparticles of heavy materials such as gold can be used as markers in quantitative electron microscopic studies of protein distributions in cells with nanometer spatial resolution. Studying nanoparticles within the context of cells is also relevant for nanotoxicological research. Here, we report a method to quantify the locations and the number of nanoparticles, and of clusters of nanoparticles inside whole eukaryotic cells in three dimensions using scanning transmission electron microscopy (STEM) tomography. Whole-mount fixed cellular samples were prepared, avoiding sectioning or slicing. The level of membrane staining was kept much lower than is common practice in transmission electron microscopy (TEM), such that the nanoparticles could be detected throughout the entire cellular thickness. Tilt-series were recorded with a limited tilt-range of 80° thereby preventing excessive beam broadening occurring at higher tilt angles. The 3D locations of the nanoparticles were nevertheless determined with high precision using computation. The obtained information differed from that obtained with conventional TEM tomography data since the nanoparticles were highlighted while only faint contrast was obtained on the cellular material. Similar as in fluorescence microscopy, a particular set of labels can be studied. This method was applied to study the fate of sequentially up-taken low-density lipoprotein (LDL) conjugated to gold nanoparticles in macrophages. Analysis of a 3D reconstruction revealed that newly up-taken LDL-gold was delivered to lysosomes containing previously up-taken LDL-gold thereby forming onion-like clusters.     

Microbial Synthesis of Antiviral Nucleosides Using Escherichia coli BL21 as Biocatalyst

Experimental conditions such as shaking (aeration) rate, concentration of reagents and extent of culture growth for the optimal synthesis of adenosine using Escherichia coli BL21 as biocatalyst were assessed, achieving 95% yield in 30 min of reaction using microorganisms harvested from late exponential phase. The ability of E. coli BL21 to synthesise purine nucleosides containing sugar residues such as 2'-deoxyribose, 2',3'-dideoxyribose and arabinose was also verified. 2'-Deoxyribo- and arabinonucleosides could be prepared in high yield, while the results obtained with 2',3'-dideoxyribonucleosides were not satisfactory. In the case of 2'-deoxyadenosine, using thymidine as a starting material, a yield of 94% was achieved at 45°C.