The Genus Gluconobacter Oxydans: Comprehensive Overview of Biochemistry and Biotechnological Applications

ABSTRACT

The genus Gluconobacter comprises some of the most frequently used microorganisms when it comes to biotechnological applications. Not only has it been involved in “historical” production processes, such as vinegar production, but in the last decades many bioconversion routes for special and rare sugars involving Gluconobacter have been developed. Among the most recent are the biotransformations involved in the production of L-ribose and miglitol, both very promising pharmaceutical lead molecules. Most of these processes make use of Gluconobacter's membrane-bound polyol dehydrogenases. However, recently other enzymes have also caught the eye of industrial biotechnology. Among them are dextran dextrinase, capable of transglucosylating substrate molecules, and intracellular NAD-dependent polyol dehydrogenases, of interest for co-enzyme regeneration. As such, Gluconobacter is an important industrial microbial strain, but it also finds use in other fields of biotechnology, such as biosensor-technology. This review aims to give an overview of the myriad of applications for Gluconobacter, with a special focus on some recent developments.     

The genus Gluconobacter and its applications in biotechnology

Organisms of the genus Gluconobacter have been widely utilized within the biotechnology industry for many decades, due to their unique metabolic characteristics. The metabolic features that render Gluconobacter so useful in biotransformation processes, vitamin synthesis, and, as the biological element in sensor systems, are critically evaluated, and the relevance of recent biochemical genetic studies to current and future industrial Gluconobacter processes is discussed. The impact of recombinant gene technology on the status of Gluconobacter processes and the potential use of such techniques in clarifying aspects of the physiology of Gluconobacter is reviewed.     

Gluconobacter oxydans: its biotechnological applications

Gluconobacter oxydans is a gram-negative bacterium belonging to the family Acetobacteraceae. G. oxydans is an obligate aerobe, having a respiratory type of metabolism using oxygen as the terminal electron acceptor. Gluconobacter strains flourish in sugary niches e.g. ripe grapes, apples, dates, garden soil, baker's soil, honeybees, fruit, cider, beer, wine. Gluconobacter strains are non-pathogenic towards man and other animals but are capable of causing bacterial rot of apples and pears accompanied by various shades of browning. Several soluble and particulate polyol dehydrogenases have been described. The organism brings about the incomplete oxidation of sugars, alcohols and acids. Incomplete oxidation leads to nearly quantitative yields of the oxidation products making G. oxydans important for industrial use. Gluconobacter strains can be used industrially to produce L-sorbose from D-sorbitol; D-gluconic acid, 5-keto- and 2-ketogluconic acids from D-glucose; and dihydroxyacetone from glycerol. It is primarily known as a ketogenic bacterium due to 2,5-diketogluconic acid formation from D-glucose. Extensive fermentation studies have been performed to characterize its direct glucose oxidation, sorbitol oxidation, and glycerol oxidation. The enzymes involved have been purified and characterized, and molecular studies have been performed to understand these processes at the molecular level. Its possible application in biosensor technology has also been worked out. Several workers have explained its basic and applied aspects. In the present paper, its different biotechnological applications, basic biochemistry and molecular biology studies are reviewed.     

Biochemistry and biotechnological applications of <Emphasis Type="Italic">Gluconobacter</Emphasis> strains

The genus Gluconobacter belongs to the group of acetic acid bacteria, which are characterized by their ability to incompletely oxidize a wide range of carbohydrates and alcohols. The corresponding products (aldehydes, ketones and organic acids) are excreted almost completely into the medium. In most cases, the reactions are catalyzed by dehydrogenases connected to the respiratory chain. Since the reactive centers of the enzymes are oriented towards the periplasmic space, transport of substrates and products into, and out of, the cell is not necessary. Thus, rapid accumulation of incompletely oxidized products in the medium is facilitated. These organisms are able to grow in highly concentrated sugar solutions and at low pH-values. High oxidation rates correlate with low biomass production, which makes Gluconobacter strains interesting organisms for industrial applications. Modern fermentation processes, such as the production of L-sorbose (vitamin C synthesis) and 6-amino- L-sorbose (synthesis of the antidiabetic drug miglitol) are carried out with members of this genus. Other important products are dihydroxyacetone, gluconate and ketogluconates. The bacteria belonging to the genus Gluconobacter exhibit extraordinary uniqueness not only in their biochemistry but also in their growth behavior and response to extreme culture conditions. This uniqueness makes them ideal organisms for microbial process development.     

Superoxide dismutase: an industrial perspective

The application of enzyme technologies to industrial research, development, and manufacturing has become a very important field. Since the production of crude rennet in 1874, several enzymes have been commercialized, and used for therapeutic, supplementary, and other applications. Recent advancements in biotechnology now allow companies to produce safer and less expensive enzymes with enhanced potency and specificity. Antioxidant enzymes are emerging as a new addition to the pool of industrial enzymes and are surpassing all other enzymes in terms of the volume of research and production. In the 1990s, an antioxidant enzyme--superoxide dismutase (SOD)--was introduced into the market. Although the enzyme initially showed great promise in therapeutic applications, it did not perform up to expectations. Consequently, its use was limited to non-drug applications in humans and drug applications in animals. This review summarizes the rise and fall of SOD at the industrial level, the reasons for this, and potential future thrust areas that need to be addressed. The review also focuses on other industrially relevant aspects of SOD such as industrial importance, enzyme engineering, production processes, and process optimization and scale-up.     

Production of foreign proteins using plastid transformation

In the past decades, the progress made in plant biotechnology has made possible the use of plants as a novel production platform for a wide range of molecules. In this context, the transformation of the plastid genome has given a huge boost to prove that plants are a promising system to produce recombinant proteins. In this review, we provide a background on plastid genetics and on the principles of this technology in higher plants. Further, we discuss the most recent biotechnological applications of plastid transformation for the production of enzymes, therapeutic proteins, antibiotics, and proteins with immunological properties. We also discuss the potential of plastid biotechnology and the novel tools developed to overcome some limitations of chloroplast transformation.     

Fumonisin and ochratoxin production in industrial Aspergillus niger strains

Aspergillus niger is perhaps the most important fungus used in biotechnology, and is also one of the most commonly encountered fungi contaminating foods and feedstuffs, and occurring in soil and indoor environments. Many of its industrial applications have been given GRAS status (generally regarded as safe). However, A. niger has the potential to produce two groups of potentially carcinogenic mycotoxins: fumonisins and ochratoxins. In this study all available industrial and many non-industrial strains of A. niger (180 strains) as well as 228 strains from 17 related black Aspergillus species were examined for mycotoxin production. None of the related 17 species of black Aspergilli produced fumonisins. Fumonisins (B(2), B(4), and B(6)) were detected in 81% of A. niger, and ochratoxin A in 17%, while 10% of the strains produced both mycotoxins. Among the industrial strains the same ratios were 83%, 33% and 26% respectively. Some of the most frequently used strains in industry NRRL 337, 3112 and 3122 produced both toxins and several strains used for citric acid production were among the best producers of fumonisins in pure agar culture. Most strains used for other biotechnological processes also produced fumonisins. Strains optimized through random mutagenesis usually maintained their mycotoxin production capability. Toxigenic strains were also able to produce the toxins on media suggested for citric acid production with most of the toxins found in the biomass, thereby questioning the use of the remaining biomass as animal feed. In conclusion it is recommended to use strains of A. niger with inactive or inactivated gene clusters for fumonisins and ochratoxins, or to choose isolates for biotechnological uses in related non-toxigenic species such as A. tubingensis, A. brasiliensis, A vadensis or A. acidus, which neither produce fumonisins nor ochratoxins.     

<Emphasis Type="Italic">Lipomyces starkeyi</Emphasis>: an emerging cell factory for production of lipids,oleochemicals and biotechnology applications

Oils and oleochemicals produced by microbial cells offer an attractive alternative to petroleum and food-crop derived oils for the production of transport fuel and oleochemicals. An emerging candidate for industrial single cell oil production is the oleaginous yeast Lipomyces starkeyi. This yeast is capable of accumulating storage lipids to concentrations greater than 60% of the dry cell weight. From the perspective of industrial biotechnology L. starkeyi is an excellent chassis for single-cell oil and oleochemical production as it can use a wide variety of carbon and nitrogen sources as feedstock. The strain has been used to produce lipids from hexose and pentose sugars derived from cellulosic hydrolysates as well as crude glycerol and even sewage sludge. L. starkeyi also produces glucanhydrolases that have a variety of industrial applications and displays potential to be employed for bioremediation. Despite its excellent properties for biotechnology applications, adoption of L. starkeyi as an industrial chassis has been hindered by the difficulty of genetically manipulating the strain. This review will highlight the industrial potential of L. starkeyi as a chassis for the production of lipids, oleochemicals and other biochemicals. Additionally, we consider progress and challenges in engineering this organism for industrial applications.     

Development of applications of industrial enzymes from Malaysian indigenous microbial sources

Malaysian enzyme industry is considered almost non-existence, although the import volume is large. Realizing the importance of enzymes, encompassing a wide range of applications in bioindustry, the development of home grown technologies for enzyme production and applications becomes one of the national priorities in industrial biotechnology. Enzyme production from indigenous microbial isolates was performed either by submerged or solid state fermentation processes. Based on its wide and unique spectrum of properties, enzymes have been developed for wide applications in various industrial processes. The development of the enzyme catalysed applications is based on the modification of the reaction systems to enhance their catalytic activities. Some of the applications of the industrial enzymes include the fine chemicals production, oleochemicals modification, detergent formulation, enzymatic drinking of waste papers, animal feed formulation and effluent treatment processes. Enzymes have also shown to be successfully used as analytical tool in the determination of compounds in body fluids. Although, most of these enzyme catalysed reactions were performed in aqueous phase, the use of enzymes in organic solvents was found to be significant for the production of new chemicals.     

New and emerging analytical techniques for marine biotechnology

Marine biotechnology is the industrial, medical or environmental application of biological resources from the sea. Since the marine environment is the most biologically and chemically diverse habitat on the planet, marine biotechnology has, in recent years delivered a growing number of major therapeutic products, industrial and environmental applications and analytical tools. These range from the use of a snail toxin to develop a pain control drug, metabolites from a sea squirt to develop an anti-cancer therapeutic, and marine enzymes to remove bacterial biofilms. In addition, well known and broadly used analytical techniques are derived from marine molecules or enzymes, including green fluorescence protein gene tagging methods and heat resistant polymerases used in the polymerase chain reaction. Advances in bacterial identification, metabolic profiling and physical handling of cells are being revolutionised by techniques such as mass spectrometric analysis of bacterial proteins. Advances in instrumentation and a combination of these physical advances with progress in proteomics and bioinformatics are accelerating our ability to harness biology for commercial gain. Single cell Raman spectroscopy and microfluidics are two emerging techniques which are also discussed elsewhere in this issue. In this review, we provide a brief survey and update of the most powerful and rapidly growing analytical techniques as used in marine biotechnology, together with some promising examples of less well known earlier stage methods which may make a bigger impact in the future.     

Characterization of membrane-bound dehydrogenases of Gluconobacter oxydans 621H using a new system for their functional expression

Acetic acid bacteria are used in biotechnology due to their ability to incompletely oxidize a great variety of carbohydrates, alcohols, and related compounds in a regio- and stereo-selective manner. These reactions are catalyzed by membrane-bound dehydrogenases (mDHs), often with a broad substrate spectrum. In this study, the promoters of six mDHs of Gluconobacter oxydans 621H were characterized. The constitutive promoter of the alcohol dehydrogenase and the glucose-repressed promoter of the inositol dehydrogenase were used to construct a shuttle vector system for the fully functional expression of mDHs in the multi-deletion strain G. oxydans BP.9 that lacks its mDHs. This system was used to express each mDH of G. oxydans 621H, in order to individually characterize the substrates, they oxidize. From 55 tested compounds, the alcohol dehydrogenase oxidized 30 substrates and the polyol dehydrogenase 25. The substrate spectrum of alcohol dehydrogenase overlapped largely with the aldehyde dehydrogenase and partially with polyol dehydrogenase. Thus, we were able to resolve the overlapping substrate spectra of the main mDHs of G. oxydans 621H. The described approach could also be used for the expression and detailed characterization of substrates used by mDHs from other acetic acid bacteria or a metagenome.

     

Membrane-bound D-sorbitol dehydrogenase of Gluconobacter suboxydans IFO 3255--enzymatic and genetic characterization

Gluconobacter strains effectively produce L-sorbose from D-sorbitol because of strong activity of the D-sorbitol dehydrogenase (SLDH). L-sorbose is one of the important intermediates in the industrial vitamin C production process. Two kinds of membrane-bound SLDHs, which consist of three subunits, were reportedly found in Gluconobacter strains [Agric. Biol. Chem. 46 (1982) 135,FEMS Microbiol. Lett. 125 (1995) 45]. We purified a one-subunit-type SLDH (80 kDa) from the membrane fraction of Gluconobacter suboxydans IFO 3255 solubilized with Triton X-100 in the presence of D-sorbitol, but the cofactor could not be identified from the purified enzyme. The SLDH was active on mannitol, glycerol and other sugar alcohols as well as on D-sorbitol to produce respective keto-aldoses. Then, the SLDH gene (sldA) was cloned and sequenced. It encodes the polypeptide of 740 residues, which contains a signal sequence of 24 residues. SLDH had 35-37% identity to those of membrane-bound quinoprotein glucose dehydrogenases (GDHs) from Escherichia coli, Gluconobacter oxydans and Acinetobacter calcoaceticus except the N-terminal hydrophobic region of GDH. Additionally, the sldB gene located just upstream of sldA was found to encode the polypeptide consisting of 126 very hydrophobic residues that is similar to the one-sixth N-terminal region of the GDH. Development of the SLDH activity in E. coli required co-expression of the sldA and sldB genes and the presence of PQQ. The sldA gene disruptant showed undetectable oxidation activities on D-sorbitol in growing culture, and resting-cell reaction (pH 4.5 and 7); in addition, they showed undetectable activities on D-mannitol and glycerol. The disruption of the sldB gene by a gene cassette with a downward promoter to express the sldA gene resulted in formation of a larger size of the SLDH protein and in undetectable oxidation of the polyols. In conclusion, the SLDH of the strain 3255 functions as the main polyol dehydrogenase in vivo. The sldB polypeptide possibly has a chaperone-like function to process the SLDH polypeptide into a mature and active form.     

Bioactive molecules from protists: Perspectives in biotechnology

For hundreds of years, mankind has benefited from the natural metabolic processes of microorganisms to obtain basic products such as fermented foods and alcoholic beverages. More recently, microorganisms have been exploited for the production of antibiotics, vitamins and enzymes to be used in medicine and chemical industries. Additionally, several modern drugs, including those for cancer therapy, are natural products or their derivatives. Protists are a still underexplored source of natural products potentially of interest for biotechnological and biomedical applications. This paper focuses on some examples of bioactive molecules from protists and associated bacteria and their possible use in biotechnology.     

葡糖杆菌属分类及其主要应用的研究进展

葡糖杆菌是醋酸菌科的一个重要属,与人类关系密切,该属中部分菌株在维生素C、米格列醇的工业生产及合成中起着重要作用;此外,该属菌株还能氧化葡萄糖生成葡糖酸盐和酮基葡糖酸等工业重要中间体。综述了葡糖杆菌的研究进展,主要介绍了葡糖杆菌的生理生化特征、分类进展及其主要应用,可以为广大醋酸菌研究者提供参考。     

Prospecting for biocatalysts and drugs in the genomes of non-cultured microorganisms

Modern biotechnology has a steadily increasing demand for vitamins, antibiotics and, in particular, novel biocatalysts for use in the production of flavors, agrochemicals, pharmaceuticals and high-value fine chemicals. Novel experimental approaches are being developed in attempts to identify such molecules. However, it is known that up to 99.8% of the microbes present in many environments are not readily culturable; hence, they cannot be exploited for biotechnology. The 'metagenome technology' offers a solution to this problem by developing culture-independent methods to isolate, clone and express environmental DNA. So far, metagenome-based approaches have led to the isolation of many novel biocatalysts and a variety of other molecules with a high potential for downstream applications.     

The Life Cycle of Chlorine, Part III

In the two previous articles in this series we reviewed the major processes of chlorine production (Part I) and its intermediate uses and waste products in the production of other chemicals (Part 11). In this article I consider some of the final applications of chlorine (e.g., for water treatment and pulp bleaching) and the uses of the most important chlorinated compounds such as solvents, chlorofluorocarbons, and the plastic polyvinyl chloride in the industrial economy. I summarize known evidence regarding their environmental fates. The special case of persistent long-lived toxic compounds (e.g., pesticides) will be discussed in a subsequent article.     

Biotechnology and fish culture

Biotechnology can currently be considered of importance in aquaculture. The increase in the production of aquatic organisms over the last two decades through the use of biotechnology indicates that in a few generations biotechnology may overtake conventional techniques, at least for the commercially more valuable species. In the last few years, genetics has contributed greatly to fish culture through the application of the more recent techniques developed in biotechnology and in genetic engineering. At present, the most commonly used methods in fish biotechnology are chromosome manipulation and hormonal treatments, which can be used to produce triploid, tetraploid, haploid, gynogenetic and androgenetic fish. These result in the production of individuals and lineages of sterile, monosex or highly endogamic fish. The use of such strategies in fish culture has as a practical objective the control of precocious sexual maturation in certain species; other uses are the production of larger specimens by control of the reproductive process and the attainment of monosex lines containing only those individuals of greater commercial value. The use of new technologies, such as those involved in gene transfer in many species, can result in modified individuals of great interest to aquaculturists and play important roles in specific programmes of fish production in the near future.