De novo fermentation

De novo fermentation has long been the method of choice for the manufacture of many natural L-amino acids, such as glutamic acid and lysine, and hydroxy acids such as lactic and citric acids. More recently, de novo fermentation is displacing existing multistep chemical syntheses, for example in the manufacture of vitamin B2 (riboflavin) and vitamin C. Other recent successes of white... 

L-Tryptophan (L-Trp) was produced, mainly by Japanese companies, on a scale of 500-6001 a-1 in 1997 [70]. It is an essential amino acid that is used as a food and feed additive and in medical applications. L-Trp is, at US 50 kg-1 (feed quality), the most expensive aromatic amino acid and it is thought that the market for L-Trp could expand drastically if the production costs could be brought down. There is no chemical process for L-TrP and enzymatic procedures starting from indole, which were very efficient, could not compete with fermentation [95]. L-Trp has been produced by precursor fermentation of anthranilic acid (ANT, see Fig. 8.16), but the serious effects of minor by-products caused the process to be closed down. Since the mid-1990s all L-Trp is produced by de novo fermentation. 

Attempts at de novo fermentation of (R)-pantothenate in organisms such as E. coli [117], Bacillus species [118] and C. glutamicum [113, 119] met with low production levels of 1-2 g L-1 [113], which is too low to be of immediate practical value. 

De-novo fermentation of ASA in an acid-tolerant yeast, combined with in-pro-cess product removal, has the potential of eliminating salt production altogether. Unfortunately, there is no microbe that naturally produces ASA and the reported yields are minute until now [176]. 

After many years of seeming stagnation, the prospects are now good that the chemical process for ASA will be swept from the market by greener, more efficient and cheaper to operate procedures based on biotechnology. The ultimate objective, de-novo fermentation from Glc, is still elusive but there is little doubt that competition between the producers eventually will cause one to be developed. 

A biotransformation, as defined by Straathof et al., ° is a process that describes a reaction or a set of simultaneous reactions in which a pre-formed precursor molecule is converted using enzymes and/or whole cells, or combinations thereof, either free or immobilised . Fermentation processes, with de novo product formation from a carbon and energy source, such as glucose via primary metabolism, are outside the scope of this chapter and book unless employed in conjunction with a biotransformation. 

The de novo synthesis of fatty acids in the mammary gland utilizes mainly acetate and some (3-hydroxybutyrate. These precursors arise from the microbial fermentation of cellulose and related materials in the rumen. Once in the mammary gland, acetate is activated to acetyl-CoA. The mechanism of fatty acid synthesis essentially involves the carboxylation of acetyl-CoA to malonyl-CoA, which is then used in a step-wise chain elongation process. This leads to a series of short-chain and medium-chain length fatty acids, which differ by two CH2 groups (e.g., 4 0, 6 0, 8 0, etc.) (Hawke and Taylor, 1995). These are straight-chain, even-numbered carbon fatty acids. However, if a precursor such as propionate, valerate or isobutyrate, rather than acetate, is used, branched-chain or odd-numbered carbon fatty acids are synthesised (Jenkins, 1993 see Chapter 2). 

The misincorporation of norleucine for methionine was known to occur in bacteria when high level synthesis of recombinant proteins were induced in minimal medium fermentation (1,2). This misincorporation was detected in the production of N-labeled recombinant human leptin produced using minimal medium conditions, however, is not present in the clinical samples produced using other fermentation conditions. The mechanism for the misincorporation was believed to involve the de novo synthesized norleucine which bypasses the leucine biosynthetic pathway and enters directly into the... 

The antibiotic thiolactomycin (43), a fermentation product from a Nocardia species containing an unusual thiolactone moiety was patented as antibiotic no. 2-200 and subsequently reported in the literature in 1982 [73,74]. It resembles a sugar-derived a,/3-unsaturated 4-thioglycono-1,4-lactone and was found to be a broad-spectmm antibiotic [75] by interference with the fatty acid metabolism of bacteria and also inhibited inducible /3-lactamases [76]. A de novo synthesis of the racemate was reported by a Du Pont group in 1984 [77]. Chambers and Thomas [78] reported the synthesis of the (55j-enantiomer in 1989 and concluded from its optical rotation that the natural product is the (5i )-enantiomer. 

There are two principal ways for utilization of microorganisms (yeasts, fungi, bacteria) for the production of flavouring substances, i.e. fermentation (de novo biosynthesis) and biotransformation (Tab. 3.7). Fermentation products are usually complex (see 3.2.2.4.). Nevertheless, there are some single flavouring substances that are produced by fermentation, such as acetic, butyric, and propionic acids and others (Tab. 3.8). For biotransformations by microorganisms, suitable substrates are necessary. Some examples are given in Tab. 3.9 and Fig. 3.5. 

Table 3.8 Examples of de novo biosynthesis of flavouring substances by microbial fermentation [20, 28-36]...

The other strategy mentioned above is the biosynthesis, isolation and purification of individual flavor-active species. This approach involves exploiting specific bio-conversions, such as oxidation and reduction or de novo syntheses by either microbial fermentation or by using specific enzyme systems. 

Industrial fermentations are generally more rapid and efficient when these materials are used, since they reduce the number of compounds which the cells would otherwise have to synthesize de novo . " The availability of nitrogen as well as the concentration in the media has to be considered in each case. Proteins can only be assimilated by microorganisms that secrete extracellular proteases, which enzymatically hydrolyze the proteins to amino acids. Microorganisms without this ability require protein hydrolysates, peptones, or digests composed of free amino acids prepared by hydrolyzing proteinaceous materials with acids or enzymes. 

The generation of individual flavor compounds or complex flavor mixrnres by the use of microorganisms and enzymes, the topic of subsequent symposium contributions, is summarized. The two basic approaches, de novo-synthesis in the course of microbial fermentations and biotransformations of suitable precursors are outlined. The increased compositional and strucmral knowledge of the substrates/precursors needed and the tailor-made design of the microorganisms/enzymes employed are presented as bases for strategies to optimize the biogeneration of flavors. 

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