Microorganism Selection And Development

Microorganisms are the biocatalysts that produce and maintain a host of enzymatic pathways that are used to produce the food component of interest. The characteristics of a good industrial microorganism for the production of food ingredients are (1) it must be effective in producing large quantities of a single product, (2) it can be efficiently isolated and purified, (3) it is easy to maintain and cultivate, (4) it is genetically stable, (5) it grows best in an inexpensive culture medium, and (6) it is safe for human consumption. The first step is to isolate the hardiest starter culture possible, then to begin strain improvements via classical mutagenesis or genetic engineering.

A classic example would be the production of L-phenylalanine for the artificial sweetener aspartame (NutraSweet® J.W. Childs Equity Partners II L.P.), which is a dipep-tide of L-phenylalanine and L-aspartic acid. When NutraSweet first entered the market in 1981, the L-phenylalanine supply became the bottleneck for production. L-Phenylalanine, L-tyrosine, and L-tryptophan are produced via the shikimic acid pathway in all organisms. To develop a bacterium which over produced L-phenylalanine, first classical chemical mutagenesis of an L-tyrosine auxotroph of Corynebacterium glutamicum was employed using -phenylalanine analog resistance in an effort to reduce end product inhibition, and L-tyrosine production (5). Analogs such as p-aminophenylalanine, p- and m-fluorophenyl-alanine, and ^-2-thienylalanine were incorporated into the cellular protein thus poisoning the cell. To combat this poison, surviving mutants must overproduce L-phenylalanine, thus neutralizing the toxic effects of the analogs. This process was repeated several times with mutants resistant to increasing concentrations of analogs. The final analog resistant bacterium selected by Hagino and Nakayama (5) produced 9.5 g/L of L-phenylalanine.

The over producing bacterium was then transformed with plasmids containing L-phenylalanine analog resistant chorismate mutase and prephenate dehydratase genes (6). These are two key enzymes in the shikimic acid pathway for L-phenylalanine production. Except for constitutive enzymes, most enzymes in the cell have a short half life in the cell. Thus, an increase in key enzyme concentrations and residence time in the cell will also increase production. Ozaki et al. (6) transformates produced 19.0 g/L of -Lphenylalanine, thus, illustrating how classical mutagenesis and molecular genetic techniques are employed to further enhance production of some desired metabolites for the food industry.

Another alternative method was whole bioconversion developed by Yamada et al. (7) which produced L-phenylalanine from trans-cinnamic acid via L-phenylalanine ammonia lyase (PAL) reversal in Rhodotorula glutinis. In the presence of concentrated ammonium hydroxide, the PAL reversal demonstrated a 70% conversion yield which produced 17.5 g/L of L-phenylalanine. By utilizing a whole cell bioconversion process, no enzyme purification step was needed and the enzyme proved to be more stable within the yeast under the harsh conditions employed. This process was used to produce some of the initial L-phenylalanine used for the production of NutraSweet. Eventually, however, production by the genetically engineered bacterium exceeded the levels in fed-batch fermentation, which did not involve caustic chemicals, and thus, became the method of choice. L-Phenylalanine purification is performed by ion exchange chromatography for all methods.

The genetic stability of cultures requires minimum culture transfers and long term storage capabilities. Fermentation media are inoculated from working cultures which are produced every few months from master cultures depending on the microorganism. The most common procedures for long term storage are freeze drying (< —18°C) and ultra-low temperature storage (—70 - —80°C). Freeze drying requires a cryoprotectant, such as sterile skim milk, followed by freeze drying and vial sealing under a vacuum (2). Sealing under nitrogen gas can also help to stabilize the culture and extend the shelf life. Ultra-low temperature storage is in a rich culture medium with 20% sterile glycerol. Some cultures are sensitive to freeze drying, thus, ultra-low is the most common method employed today, because of long-term culture viability. The risk is loss of electrical power and refrigeration problems.

Suspended cell cultures or spore suspensions are used as inocula for these industrial scale fermentations. Purity is constantly checked until inoculation. For suspended cell inoculation the sequence employed would be culture slant, shake-flask culture, benchtop fermen-tor, pilot-scale fermentation, then into full scale fermentation. Many fungal fermentations, such as citric acid and soy sauce fermentations, required a suspension of viable fungal spores as the inoculum. These spore suspensions are generated on large agar trays, and then are aseptically transferred into culture bottles suspended in sterile water or saline (2).

Microbial systems frequently constitute efficient mechanisms for the production of nutritionally important ingredients at a relatively low cost, for example, the production of selenomethione in yeast. A slight modification in the yeasts culture medium will force the yeast to substitute the sulfur group in methionine and cysteine with a selenium in standard fed-batch fermentation (8,9). To identify this medium change, Demirci and Pometto (8) developed a gradient delivery unit producing a gradient of sodium selenite or sodium sel-enate in a continuous bioreactor (Figure 6.1). It has been shown that selenium has several health benefits (10) including a cancer-protective effect (11), and a profound effect on the survival of HIV-infected patients (12).

Furthermore, microbial systems are ideal for the production of essential micronutri-ents such as amino acids, vitamins, and enzymes, and bulk ingredients such as organic acids and alcohols, whole cell flavor enhancers, and polysaccharides.

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