Bacillus species are an important source of industrial enzymes, fine biochemicals, antibiotics, and insecticides (5). Additionally, the ability of B. subtilis and close relatives to secrete grams per liter quantities of protein directly into the growth medium, their ease of growth, and their well-proven safety have also made them prime candidates for the production of heterologous proteins.
Many commercially important enzymes have traditionally been produced in their native hosts. However, when organisms with novel enzymic activities are newly isolated, the costs and uncertain outcome associated with process optimization are often prohibitive. This even applies to well-established strains of B. amyloliquefaciens and B. licheniformis that have been used extensively in commercial enzyme fermentations. Both these organisms are generally refactory to genetic engineering, and relatively little information is available concerning their biochemistry and physiology. Instead, companies are increasingly seeking to develop well-characterized strains such as B. subtilis strain 168 as universal hosts for commercial applications.
The world annual sales for industrial enzymes was recently valued at $1 billion, with strong growth in the paper, textiles, and waste treatment markets. Three-quarters of the market is for enzymes involved in the hydrolysis of natural polymers, including proteolytic enzymes used in the detergents, dairy, and leather industries and carbo-hydrases used in the baking, brewing, distilling, starch, and textile industries. About two-thirds of these enzymes are produced by fermentation from Bacillus species, the main producers, together with their products and activities, are listed in Table 1.
Two Bacillus enzymes dominate the industrial enzymes markets alkaline (serine) protease and a-amylase. Although many species of Bacillus secrete enzymes of these types, their catalytic properties vary from one producer strain to the next. Moreover, commercially significant enzymes may be modified to improve their performance or stability in particular industrial processes.
Alkaline proteases are the single largest enzyme market and are used extensively as additives to detergents. They are relatively nonspecific endoproteases that convert substrate proteins into small, readily soluble fragments. The two main proprietary products, subtilisin Novo (BPN') from B. amyloliquefaciens and subtilisin Carlsberg from B. licheniformis, have been the subject of extensive develop-
Table 1. Commercially Significant Products Produced by Bacillus species
Glucose isomerase a-Amylase j?-Amylase y?-Glucanase
Pullulanase Alkaline protease
Glucose dehydrogenase Glycerol kinase Phospholipase C Restriction endonucleases
¿-Endotoxin Peptide antibiotics
B. amyloliquefaciens B. stearothermophilus B. licheniformis B. polymyxa B. amyloliquefaciens B. circulans B. subtilis Bacillus species B. alkalophilus B. licheniformis B. thermoproteolyticus B. thermoproteolyticus B. stearothermophilus B. subtilis B. mesenterius B. cereus B. polymyxa B. megaterium B. licheniformis B. megaterium B. stearothermophilus B. cereus
B. aneurinolyticus B. amyloliquefaciens B. subtilis B. caldolyticus B. globiii
B. stearothermophilus B. sphaericus B. brevis B. thuringiensis B. licheniformis B brevis B. subtilis B. polymyxa
Conversion of glucose to fructose in manufacture of low calorie sweeteners
Limited hydrolysis of starch
Liquefication of starch
Thermophilic liquefaction of starch
Thermostable liquefaction of starch
Formation of low-dextrose-equivalent syrups from starch Hydrolysis of barley y?-glucans in brewing and animal feeds
1,6-Bond starch debranching enzyme Detergents and leather industries
Condensation of l-aspartic acid and d,l-phenylalanine to produce aspartame Dairy industry
Biocontrol agent Bacitracin
Edeines, gramicidin, tyrocidine Fengycin, subtilin, surfactin Polymyxins ment programs. Their three-dimensional structures have been solved (82,83), and functionality testing has resulted in the identification of the catalytic triad, an oxyanion-binding site, and substrate-binding determinants. Subtil-isins have been engineered to improve their characteristics in washing detergents by increasing their stability in the presence of oxidizing chemicals, at temperatures above 60 °C and at pH values up to 12. They have pH optima of about 11.0 and half-lives of approximately 50 min at 40 °C. They are used at 0.4-1% in formulations that includes anionic and nonanionic surfactants, oxidants, soap, and optical brighteners. Although these were originally used simply in the form of a powder, hypersensitivity reactions among producers and users meant that the enzymes had to be added to formulations in a microencapsulated form. This process not only reduces dust formation but also protects the enzymes from other components of the formulation during storage.
a-Amylases are used extensively in the starch industry where, because of the physical properties of starch, they need to be used at high temperatures and good thermosta-
bility is an important property of these enzymes. Industry has sought to obtain thermostable amylases by screening for new sources and by improving the thermostability of existing enzymes by protein engineering. For example, the a-amylases from B. amyloliquefaciens, B. stearothermophilus, and B. licheniformis are closely related (3) but have very different thermostabilities of 2, 50, and 270 min, respectively, at 90 °C and pH 6.5 (84,85). Extra salt bridges caused by specific lysine residues in the more-thermostable B. licheniformis amylase are responsible for its increased stability, and the equivalent residues in the less-stable enzymes have been the target for protein engineering. Additionally, the extensive homology between the genes encoding these amylases has also been used for in vivo recombination experiments in which hybrid enzymes combining beneficial characteristics of these enzymes have been screened. The three-dimensional structure of the B. licheniformis a-amylase has recently been published (86), and the three amino acid residues that constitute the calcium-binding site (i.e., asparagine-104, aspartate-200, and histidine-235), have been identified.
The a-amylase from B. acidocaldarius strain A2 shows greater thermostability under acid conditions (87). Additionally, B. subtilis strain KP1064 (88) produces an enzyme that is active against starch and pullulan.
The industrial production of a-amylase was developed in 1917 using a strain subsequently reclassified as B. am-yloliquefaciens. This a-amylase could be used to a maximum temperature of about 85 °C. Because starch needs to be cooked to 100 °C to rupture the starch granules, the introduction of a thermostable a-amylase from B. licheni-formis (89) that can operate at about 105 °C was a distinct improvement.
B. coagulans is an important source of glucose isomer-ases, required for the conversion of glucose (0.75 times as sweet as sucrose) to fructose (twice as sweet as sucrose) in the production of high-fructose corn syrup. In contrast to the proteases and amylases already discussed, glucose isomerase is an intracellular enzyme used either in the form of immobilized cells or extracted and immobilized on a solid matrix.
Bacillus species are used for the production of a number of primary metabolites for the food and health care industries. Extensive knowledge of their biochemistry and regulation has allowed growth and rational mutant isolation strategies to be developed to the extent that this organism has been considered for the production of a wide range of metabolites at commercially viable levels.
B. subtilis has been used for the production of the nucleotides xanthanylic acid (XMP), inosinic acid (IMP), and guanylic acid (GMP), which are of commercial importance as flavor enhancers. The primary fermentation products are the nucleosides hypoxanthine, inosine, and guanosine because their greater membrane permeability allows them to accumulate to higher concentrations in the culture medium. These are then converted to their respective nucle-otides by chemical phosphorylation (90).
Attempts have been made to develop strains of Bacillus for the production of amino acids such as tryptophan, his-tidine, and phenylalanine and of vitamins such as biotin, folic acid, and riboflavin. The use of analogs has led to the isolation of resistance mutants overproducing a number of amino acids, nucleosides, and vitamins (91). Knowledge of B. subtilis is such that rational approaches are now possible and attempts have been made to engineer this bacterium for the production of folic acid. Promising strains have been obtained, although their production levels cannot yet compete with those of Brevibacterium, Corynebac-terium, or Serratia species.
Under conditions of nutritional stress Bacillus species produce a variety of special metabolites that enhance their survival in natural environments (92). Prominent among these are peptide antibiotics, generally short peptides (220 amino acid residues) that are synthesized by large multienzyme complexes (peptide synthetases) rather than ri-bosomes. Individual amino acid residues are often extensively modified and may be linked to each other by peptide bonds or through esters or lactones. Gramicidin-S is a cyclic decapeptide from B. brevis with antibacterial and surfactant properties. Bacitracin is a branched cyclic dodecapeptide produced by B. licheniformis and B. subtilis
(93) that is used as a topical antibiotic. Its antibacterial activity is directed against bacterial cell wall synthesis where it inhibits the recycling of lipid-P carriers. Surfactin, produced by most strains of B. subtilis, has both antibacterial activity and powerful surfactant properties. Poly-myxins are membrane-active branched cyclic acylpeptides produced by B. polymyxa.
A minority of the peptide antibiotics produced in Bacillus species are synthesized on ribosomes and subsequently modified extensively by posttranslational processing (92). The resulting products are usually larger, but only marginally so, than those produced by the peptide synthetases. The best studied are the lantibiotics, so-called because they contain the unusual amino acids lanthionine and methyl-lanthionine. They include subtilin, a 32-residue lantibiotic produced by B. subtilis that shows antibacterial and antitumor activity.
The efficient large-scale production of heterologous enzymes and proteins by B. subtilis has been the focus of considerable research effort. The ability of this bacterium to secrete proteins directly into the culture medium in quantities of grams per liter offers considerable process advantages, in terms of product yield, structural integrity, and downstream processing costs, over production systems in which proteins are accumulated in the cytoplasm or periplasm, often as insoluble aggregates.
Nevertheless, attempts to use B. subtilis for the production of heterologous proteins has met with only limited success, and there are no reports of the use of this host in an industrial fermentation. Although extracellular proteins from close relatives can be produced in this bacterium at high concentrations, the yield of proteins from unrelated species remains disappointingly low. This is likely to be due, at least in part, to the production of at least seven extracellular proteases, which have been shown to cause extensive degradation of heterologous proteins, and to incompatibilities with the Bacillus protein secretion pathway. The isolation of strains defective in the identified proteases has helped in some but by no means all cases (61,63).
Other species of Bacillus may prove useful for development as hosts for the production of heterologous proteins, particularly those naturally producing low levels of extracellular proteases. Of interest are species such as B. brevis that produce a crystalline surface (S) layer at the outer surface of a thin cell wall. S-layer proteins have been observed at concentrations up to 35 g/L in the culture medium and, consequently, attempts have been made to develop the expression and signal sequences of their genes into components of a new generation of secretion vectors
(94). However, it may ultimately be necessary to develop strains that are currently used in industrial processes as production hosts by improving our understanding of their genetics and physiology.
B. subtilis has also proved useful for the intracellular production of outer membrane proteins of gram-negative pathogens (95). These proteins, which have potential for use for immunodiagnostics and as vaccines, are produced in B. subtilis in preference to their native hosts to avoid contamination with endotoxins with which they often form tight associations.
The use of chemical insecticides, with a world market worth $5 billion, is increasingly seen as problematical because of the development of resistance in target insect populations, lack of specificity against the insect pest, persistence in the environment, and toxicity to man and other animals. Alternative biocontrol methods have been developed involving the use of viruses, fungi, and bacteria. B. thuringiensis (96) is the most successful insecticidal biocontrol agent and accounts for about 90% of the worldwide market for bioinsecticides. Although B. thuringiensis was originally isolated in 1901 from an infection of a Japanese silkworm farm and has been used as an insecticidal agent since 1920, it still only represents about 1-2% of the total insecticide market.
Strains of B. thuringiensis have been identified that are pathogenic for each of the main groups of insect pests, namely Lepidotera (moths, butterflies), Diptera (flies and mosquitoes), and Coleoptera (beetles). More recently strains of B. thuringiensis have been isolated that are active against noninsect pests, notably nematodes, mites, and protozoa (97). The toxicity of B. thuringiensis results from the production of proteinaceous ¿-endotoxins during sporulation. The toxins, which may represent as much as 30% of the cell's weight, form crystals within the mother cell (98). The toxins are synthesized from plasmid-encoded cry genes, and a single strain may produce several toxins encoded by more than one plasmid. They are synthesized initially in the form of an inactive pro-toxin (70-140 kDa) that is considerably larger than the active form (60 kDa). The ¿-endotoxins, which have the properties of chemical agents rather than infectious agents, are approximately 300 times more potent than pyrethroids and 8,000 times more potent than organophosphates. B. thuringiensis toxins therefore combine high toxicity and specificity for their target pests with little or no toxicity for nontarget insects (e.g., pollinators, predators) and other animals.
A large number of cry genes have been cloned, and the regions responsible for the pro- and mature forms, host specificity, and toxicity have been mapped. As different toxins exhibit a range of host specificities and levels of toxicity, chimeric toxins that combine the specificities and toxic regions of different natural toxins are currently being developed.
B. thuringiensis strains have been used extensively for the control of crop and forest insect pests and of insect disease vectors. However, as an alternative means of application, genes encoding the ¿-endotoxin have been used directly to transform a variety of plant crops, including tomatoes, tobacco, potatoes, and cotton, in which field trials have confirmed their activity against target pests (99).
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