Directed evolution of biocatalytic processes

The benefits of applying biocatalysts to organic synthesis, such as their high chemo-, regio-, and enantio-specificity and selectivity, must be seriously considered, especially where chemical routes are unavailable, complex or prohibitively expensive. In cases where a potential biocatalytic route is not yet efficient enough to compete with chemical synthesis, directed evolution, and/or process engineering could be implemented for improvements. While directed evolution has demonstrated great potential to enhance enzyme properties, there will always be some aspects of biocatalytic processes that it does not address. Even where it can be successfully applied, the resources required for its implementation must currently be weighed against the feasibility of, and resources available for developing a chemical synthesis route. Here, we review the potential of combining directed evolution with process engineering, and recent developments to improve their implementation. Favourable targets for the directed evolution of new biocatalysts are the syntheses of highly complex molecules, especially where chemistry, metabolic engineering or recombineering provide a partial solution. We also review some of the recent advances in the application of these approaches alongside the directed evolution of biocatalysts.     

Application of radiolabeled tracers to biocatalytic flux analysis.

Radiolabeled tracers can provide valuable information about the structure of and flux distributions in biocatalytic reaction networks. This method derives from prior studies of glucose metabolism in mammalian systems and is implemented by pulsing a culture with a radiolabeled metabolite that can be transported into the cells and subsequently measuring the radioactivity of all network metabolites following separation by liquid chromatography. Intracellular fluxes can be directly determined from the transient radioactivity count data by tracking the depletion of the radiolabeled metabolite and/or the accompanying accumulation of any products formed. This technique differs from previous methods in that it is applied within a systems approach to the problem of flux determination. It has been used for the investigation of the indene bioconversion network expressed in Rhodococcus sp. KY1. Flux estimates obtained by radioactive tracers were confirmed by macroscopic metabolite balancing and showed that indene oxidation in steady state chemostat cultures proceeds primarily through a monooxygenase activity forming (1S,2R)-indan oxide, with no dehydrogenation of trans-(1R,2R)-indandiol. These results confirmed the significance of indan oxide formation and identified the hydrolysis of indan oxide as a key step in maximizing the production of (2R)-indandiol, a chiral precursor of the HIV protease inhibitor, Crixivan.     

Terpene glucoside production: Improved biocatalytic processes using glycosyltransferases

Terpenoids are one of the main classes of natural products. In plants, a large fraction of the terpenoids is present as nonvolatile glycosides. The terpene glycosides have attracted much attention as antimicrobials, flavor precursors, and detergents. They are either extracted from plant materials or are synthesized by chemical and biocatalytic methods. Up to now, biotechnological production of terpene glycosides is based on reversed hydrolysis performed by glycosidases. However, this method suffers from low yields as a matter of principle. Recently, the first uridine diphosphate‐glucose:monoterpenol β‐d ‐glucosyltransferase (GT) genes were cloned and characterized from grapevine (Vitis vinifera) and kiwi (Actinidia deliciosa). Heterologous expression in Escherichia coli yielded promiscuous GT enzymes that efficiently glucosylated primary monoterpenols, simple alcohols, and phenols. The GT enzymes differed in substrate preference and activity toward their terpenoid substrates. Biotransformation experiments confirmed the applicability of the novel GTs in biocatalytic processes for the production of these novel compounds. In the near future, terpene glucosides will become commercially available for food, cosmetic, and pharmaceutical industry due to improved biocatalytic processes involving GT enzymes.     

Comparison of techniques permitting to detect apoptosis in situ

Several morphological and biochemical techniques are in use for identification of apoptotic and necrotic cells in a studied cell population. It is essential to define not only the type of cell death but also to identify the apoptotic process itself, which represents a multistage, active process, requiring activation of a molecular event cascade. In the present study, we have examined and discussed effectiveness of the selected techniques detecting apoptosis in lymphocytes exposed to incubation at an elevated temperature. The appraisal involved detection of caspase-3 active form, detection of Bcl-2, TUNEL reaction, the comet assay and electrophoresis of DNA.     

Application of in situ hybridization, cytochemical and immunocytochemical techniques for the investigation of peroxisomes

 In situ hybridization, cytochemical and immunocytochemical techniques have contributed significantly to the understanding of the biology of peroxisomes, since they permit in situ demonstration of the sites of synthesis and distribution of peroxisomal proteins without the necessity of homogenization and subcellular fractionation of tissues or cultured cells. This article reviews the results of research on mammalian peroxisomal metabolism, biogenesis and proliferation in which morphological techniques have played a significant role in the elucidation of the biological problem. Some new data on peroxisomal heterogeneity and morphogenesis are included. The morphological methods applied have made it possible to characterize the differences in distribution of mRNAs encoding peroxisomal proteins in different tissues, as well as to monitor the marked heterogeneity in the protein composition and in the activity of specific enzymes in the peroxisomal population of single cells, or in tissues with complex organization (e.g. liver and kidney). In addition, the dynamic alterations and high plasticity of the peroxisomal compartment – partly dependent on contact of the peroxisomes to the microtubular network – are presented.     

Application of in situ PCR to yeast cells for screening YAC libraries.

We have used in situ PCR technology in yeast cells with the ultimate goal of cloning and screening genomic yeast artificial chromosome (YAC) libraries. The target sequences in YAC clones were amplified "in situ" in yeast cells by the same set of microsatellite primers used in solution-based PCR screening. The method is fast and sensitive and obviates the steps required for individual isolation of DNAs from hundreds to thousands of YAC clones and thus has an advantage over conventional solution-based PCR screening. This approach can conceivably be applied to the products of automated robotic workstations.     

Application of artificial neural network coupling particle swarm optimization algorithm to biocatalytic production of GABA

The biotransformation of L-sodium glutamate (L-MSG) to gamma-aminobutyric acid (GABA) catalyzed by the cells of Lactobacillus brevis with higher glutamate decarboxylase activity was investigated. The results showed that pH, temperature, and FeSO(4) x 7H(2)O concentration had significantly positive effect on GABA yield. The individual and interactive effects of pH, temperature, and FeSO(4) x 7H(2)O concentration were further optimized in terms of GABA yield. In the present work, an artificial neural network (ANN) and response surface methodology (RSM) models were developed, which incorporated pH, temperature, and FeSO(4) x 7H(2)O concentration as input variables, and GABA yield as output variable. The optimized ANN topology included four neurons in the hidden layer and the best network architecture was 3-4-1. The trained ANN gave total root-mean square error (sigma) equal to 1.84 for GABA yield while the RSM gave sigma equal to 2.63. The results demonstrated a slightly higher prediction accuracy of ANN compared to RSM. The modeled maximum GABA yield was identified by applying particle swarm optimization algorithm to the ANN model developed. The modeled maximum GABA yield reached 91 mM under the following optimal conditions: 25 mL Na(2)HPO(4)-citric acid buffer (100 mM, pH 4.23), 120 mM L-MSG, 0.83 g/L FeSO(4) x 7H(2)O, 10 microM PLP, the resting cells obtained from a 60-h culture broth, 2.68 g dry cell weight (DCW)/L, and without agitation at 40 degrees C for 5 h. The previous high value of GABA yield that was observed was 81.8 mM. The optimized conditions allowed GABA yield to be increased from 81.8 to 90.57 mM after verification experiments test.     

In situ sensor techniques in modern bioprocess monitoring

New reactor concepts as multi-parallel screening systems or disposable bioreactor systems for decentralized and reproducible production increase the need for new and easy applicable sensor technologies to access data for process control. These sophisticated reactor systems require sensors to work with the lowest sampling volumes or, even better, to measure directly in situ, but in situ sensors are directly incorporated into a reactor or fermenter within the sterility barrier and have therefore to stand the sterilization procedures. Consequently, these in situ sensor technologies should enable the measurement of multi-analytes simultaneously online and in real-time at a low price for the robust sensing element. Current research therefore focuses on the implementation of noninvasive spectroscopic and optical technologies, and tries to employ them through fiber optics attached to disposable sensing connectors. Spectroscopic methods reach from ultraviolet to infrared and further comprising fluorescence and Raman spectroscopy. Also, optic techniques like microscopy are adapted for the direct use in bioreactor systems (Ulber et al. in Anal Bioanal Chem 376:342–348, 2003) as well as various electrochemical methods (Joo and Brown in Chem Rev 108:638–651, 2008). This review shows the variety of modern in situ sensing principles in bioprocess monitoring with emphasis on spectroscopic and optical techniques and the progress in the adaption to latest reactor concepts.     

FISH and Calcofluor staining techniques to detect in situ filamentous fungal biofilms in water

Filamentous fungi are a ubiquitous and diverse group of eukaryotic organisms and may contribute, along with bacteria, yeasts, protozoa and viruses, to the formation of biofilms in water distribution systems. However, fungal involvement in biofilms has not been demonstrated unambiguously. Furthermore, these fungi may be responsible for the production of tastes, odours and mycotoxins in drinking water making their early detection important. The detection of fme these problems a combination of two fluorescent techniques for direct detection was tested: (a) Fluorescence In Situ Hybridization (FISH) employing the universal rRNA probe EUK516, labelled with the red Cy3, followed by (b) staining with Calcofluor White MR2 fluorescent dye which stains fungal cell walls blue. Pure cultures of Penicillium brevicompactum were used to establish the methods followed by separate experiments with real water biofilm samples in PVC-C and cast iron coupons. FISH demonstrated eukaryotic microrganisms after approximately 5 h while the calcofluor method revealed chitinous filamentous structures in less than one hour. When the two methods were combined, additional resolution was obtained from the images of filamentous walls (blue) with intact protoplasm (red). In conclusion, FISH and Calcofluor staining provide rapid, direct and unambiguous information on the involvement of ff in biofilms which form in water.     

解决氧化还原酶反应体系中辅酶问题的策略及其应用

氧化还原酶还原醛或酮生成各种手性醇或手性胺类化合物。然而,氧化还原酶的催化过程通常需要价格昂贵的烟酰胺类辅酶提供或接受电子,这严重阻碍了氧化还原酶的工业化进程。因此,如何降低辅酶的成本已成为生物催化领域的研究热点和关键问题。随着工业化应用的实际需求和研究工作的深入,各种解决辅酶问题的策略被相继提出,如构建体外辅酶再生系统,利用发酵工程与代谢工程等手段提高内源性辅酶利用率,研究和开发辅酶替代物等。文中对这些策略的研究概况进行简要介绍,并通过列举相关应用实例分析各自的优、缺点,为进一步拓展氧化还原酶的工业化应用提供借鉴和参考。     

Radioactive phosphorylation of alcohols to monitor biocatalytic Diels-Alder reactions

Nature has efficiently adopted phosphorylation for numerous biological key processes, spanning from cell signaling to energy storage and transmission. For the bioorganic chemist the number of possible ways to attach a single phosphate for radioactive labeling is surprisingly small. Here we describe a very simple and fast one-pot synthesis to phosphorylate an alcohol with phosphoric acid using trichloroacetonitrile as activating agent. Using this procedure, we efficiently attached the radioactive phosphorus isotope (32)P to an anthracene diene, which is a substrate for the Diels-Alderase ribozyme-an RNA sequence that catalyzes the eponymous reaction. We used the (32)P-substrate for the measurement of RNA-catalyzed reaction kinetics of several dye-labeled ribozyme variants for which precise optical activity determination (UV/vis, fluorescence) failed due to interference of the attached dyes. The reaction kinetics were analyzed by thin-layer chromatographic separation of the (32)P-labeled reaction components and densitometric analysis of the substrate and product radioactivities, thereby allowing iterative optimization of the dye positions for future single-molecule studies. The phosphorylation strategy with trichloroacetonitrile may be applicable for labeling numerous other compounds that contain alcoholic hydroxyl groups.