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Glutamate production is a typical success in industrial fermentation. Annual production of glutamate by is over 1.5 million tons per year worldwide. It is well known that there are some triggers of glutamate overproduction by : depletion of biotin, which is required for cell growth; addition of detergent; addition of β-lactam antibiotics such as penicillin; and addition of ethambutol or cerulenin. A marked change in metabolic pathways occurs after glutamate overproduction is triggered. In this chapter, recent studies on the molecular mechanisms of glutamate production are described with a particular focus on triggering mechanisms, changes in key enzyme activities, and secretion of glutamate. Recent advances in genome-wide studies, including genomics, proteomics, metabolomics, and on metabolic flux analysis of flux redistribution during glutamate overproduction are discussed as well. The biosynthesis of the related amino acids glutamine and proline and strategies for their overproduction are also described.

-lysine is an essential amino acid required for nutrition of animals and humans. It has to be present in food and feed, which, in many cases, is realized by supplementation of the feed-stuffs with pure lysine. The high importance of lysine in nutrition has stimulated intensive research on the lysine biosynthetic pathways and their regulation and the search for microorganisms capable of over-producing this amino acid. As an important milestone, the glutamate producing soil bacterium was isolated in 1956 and soon received interest to be used for production of another amino acid stemming from the TCA cycle: lysine. Within a few years the first lysine producing strains were obtained. The past 50 years following the discovery of were characterized by a huge progress towards understanding the physiology of this organism and developing and optimizing industrial production strains. This has resulted in effective biotechnological processes currently used for producing about 750 000 tons of lysine per year. Today, systems-oriented approaches aiming at investigating the link between the different components of cellular physiology such as transcriptome, fluxome and metabolome, provide a novel powerful platform that will surely drive future research towards holistic understanding of lysine over-producing microorganisms as well as the creation of superior production strains.

This review focuses on the principles and recent progress in production of the essential amino acid -threonine. Behind glutamic acid, methionine and lysine, threonine is one of the most important amino acids almost exclusively used in the feed industry. Basic principles of threonine producers, like amplified genes coding for enzymes involved in the biosynthesis, are explained. Possible modifications of parts of the metabolism that are not directly related and a section about recent findings on global regulation round up the review of strain improvement or strain breeding. In a second section important necessities of the bioprocess industry, such as reduction of gradient formation and other scale-up related topics, are discussed. Strategies for avoiding such problems by improved reactor design or process modifications are presented and discussed in relation to recent advances in strain improvements. Finally, future steps are presented and discussed at the end of the review.

Biosynthesis of the three aromatic amino acids (-phenylalanine, -trypto phan, -tyrosine) and its regulation in and corynebacteria are reviewed. The common aromatic biosynthetic pathway (shikimate pathway) starts with the condensation of phosphoenolpyruvate and erythrose 4-phosphate. Through six biosynthetic steps the pathway proceeds via shikimate to chorismate, from which the terminal pathways to tryptophan, phenylalanine and tyrosine branch. The first step in the common pathway is performed in by three isoenzymes, which are specifically feedback-inhibited by the three terminal products.

The pathway to tryptophan starts with anthranilate formation and includes reactions with -serine and 5-phosphoribosyl-pyrophosphate. Phenylalanine and tyrosine biosyntheses proceed via prephenate, and each include a decarboxylation and transamination step. The first committed steps of each terminal pathway are strictly regulated by feedback inhibition, repression and partly through attenuation (in ).

-Tryptophan and -phenylalanine are essential amino acids for man and most livestock. Main microbial producer strains are and . Strain development includes alleviation of the various regulatory levels (feedback inhibition resistance, derepression), both in the common aromatic pathway and in the terminal pathways. In recent years, metabolic engineering has also taken into account the fact that precursor supply may become limiting once the other impediments for carbon flux are gone. Strains with improved phosphoenolpyruvate and/or erythrose 4-phosphate supply have successfully been developed. Applications for -tryptophan are the feed and pharmaceutical markets, while -phenylalanine is mainly used as building block for the artificial sweetener, aspartame

. A possible application for -tyrosine is as a building block for the synthesis of -DOPA.

The branched-chain amino acids (BCAAs) leucine, isoleucine, and valine are synthesized by bacteria, fungi, and plants, but are essential for vertebrates including humans, who must receive them from their diet. The interest to construct overproducing industrial strains therefore stems from the need to supplement the food or feed with these amino acids to use them in medical treatment and as precursors in biochemical synthesis. Regulation of the biosynthesis pathways of branched-chain amino acids has many features, such as homologous reactions catalyzed by a single enzyme, branching of the pathways and multivalent regulation of both gene expression and enzyme activity, which make their analysis both interesting and challenging. The structural similarity of these three amino acids and their precursors causes their alternative binding to the proteins as substrates, inhibitors, activators, and passengers in transporters with different affinities. Studies of threonine deaminase, the first enzyme specific for isoleucine biosynthesis, and of acetohydroxyacid synthase, the first common enzyme in the pathways of BCAA biosynthesis, promoted the discovery of feedback inhibition and may serve as paradigms for this regulatory mechanism. Regulation of the operons and provides examples of typical translation-mediated transcriptional termination (attenuation). Mechanisms of regulation by the seemingly similar structures found in operon and gene of the industrial amino acid producer still have to be unveiled. A wide range of different specific and global regulatory mechanisms being gradually uncovered in various microorganisms will contribute to the knowledge of genetic control of BCAA biosynthesis.

The sulphur-containing amino acid methionine and its derivatives play important roles in cellular metabolism. These include initiation of protein biosynthesis, methyl transfer and synthesis of polyamines. Methionine cannot be synthesized by humans and animals and must therefore be obtained from the diet. Since concentrations of methionine in plant-based diets are low, the amino acid is routinely added to animal feed. For this purpose methionine is produced in large scale by chemical synthesis. Currently, no fermentative methionine production process exists, but for economic reasons its development has recently received increasing attention. Research has mainly focused on methionine biosynthesis in the bacteria and , which are already used for the production of several other amino acids. Investigation of methionine biosynthesis and its regulation in has revealed major differences with the corresponding metabolism. This review intends to summarize the current knowledge on methionine biosynthesis in these organisms and discusses approaches for the construction of methionine producer strains.

Sulfur is necessary for the synthesis of cysteine. Microorganisms can use sulfate, thiosulfate or sulfonates as sole sulfur sources. These compounds are taken up by specific transporters followed by the conversion of sulfate or sulfonates into sulfide in 2 to 4 steps. The biosynthesis of cysteine from serine in bacteria is carried out by a two-step pathway beginning with the -acetylation of serine, followed by -re placement of the acetyl group by sulfide or thiosulfate. Some microorganisms can also use methionine or cysteine-derived compounds such as glutathione as sole sulfur source. Glutathione is degraded to liberate cysteine, whereas methionine is converted into cysteine via the reverse transsulfuration pathway or via methanethiol formation. Cysteine is also taken up directly from the environment by ABC transporters or symporters mainly as cystine, the disulfide-linked cysteine dimer. Several mechanisms are involved in the control of the intracellular concentration of cysteine, which is a highly reactive compound due to its –SH group. This amino acid is degraded mainly by cysteine desulfhydrases or is excreted by exporters. A large variety of molecular mechanisms participate in fine-tuning the regulation of cysteine metabolism: positive regulation by LysR-type regulators, negative control by repressors of the Rrf2 or TetR family and regulation by premature termination of transcription. In and , a global regulator, CysB and CymR, respectively, controls cysteine synthesis and transport in response to -acetylserine or its derivative -acetyl-serine availability. In and , a unique regulator modulates the methionine and cysteine metabolisms. Cysteine or derivative compounds are biotechnically interesting. Fermentation processes with or involving mutants insensitive to feedback inhibition by cysteine and also strains overproducing cysteine exporters or inactivated for cysteine degradative enzymes are currently being developed.

A vast number of prokaryotic and eukaryotic microorganisms can synthesize arginine de novo from glutamate. The first committed step of this pathway is acetylation of -glutamate at the -position. A surprising variety of proteins were found to catalyze this reaction in prokaryotes: (i) the classical, two-domain N-acetylglutamate synthase (NAGS) originally found in γ-Proteobacteria, (ii) shorter NAGS of the GNAT acetyltransferase family, either independent or fused with the ArgH protein (argininosuccinase), (iii) bifunctional ornithine acetyltransferases (OAT), i.e. able to acetylate glutamate with both acetyl-CoA and acetylornithine. In many organisms, including most Archaea, the enzyme acetylating glutamate remains elusive; possible connections with lysine biosynthesis may be envisaged. In fungi, NAGS appears only distantly related to its prokaryotic analog and requires association with acetylglutamate kinase (NAGK) to be functional. In most organisms, the acetyl group of acetylornithine is either split by an acetylornithinase (AO) or recycled on glutamate by OAT; in both cases, one of the products is ornithine which is carbamoylated into citrulline. In some Proteobacteria however, acetylornithine is carbamoylated into acetylcitrulline. These discoveries on arginine precursor acetylation have important metabolic and evolutionary implications.

Among Bacteria, regulation of arginine biosynthetic genes was analyzed intensively in , , and more recently in sp. Archaea remain to be investigated. Among Eukarya, the yeast was studied in great detail. The comparison of the mechanisms found to operate in these very different organisms is interesting from several points of view: (i) the occurrence of repressor-operator interactions on both sides of the prokaryote/eukaryote divide and the first evidence for a Jacob-Monod regulatory mechanism in eukaryotes, (ii) the coordination of carbamoyl phosphate synthesis with the two pathways that depend on this metabolite (arginine and pyrimidine biosyntheses), (iii) the coordination of arginine biosynthesis with arginine catabolism and the first indication ever that a repressor (the ArgR protein) may also function as a gene activator, (iv) the extensive conservation of the ArgR/AhrC transcription control system throughout the bacterial domain with the notable exception of sp. and related Bacteria, (v) the functional and possible evolutionary relationship between proteins involved in arginine metabolic control systems and proteins controlling DNA replication and partition.

Arginine is an important “nutraceutical”. Knowledge of the regulatory mechanisms controlling the function or synthesis of arginine biosynthetic enzymes in prokaryotes has been used to engineer arginine-overproducing strains amenable to industrial exploitation.

The biosynthesis of glycine and -serine is closely connected, and both amino acids are produced in industry. However, whereas glycine is made chemically, -serine production relies largely on microbial processes. These include conversions of added glycine by C-1 utilizing microorganisms. But such precursor conversions usually suffer from low yields, as did previous attempts to produce -serine from glucose. As more recent molecular and physiological studies have shown, microorganisms like have a high -serine degradation capacity corresponding to an apparent key position of this amino acid in metabolism. Considering this key position, deletion of a serine dehydratase gene and prevention of folate synthesis to reduce serine hydroxymethyltransferase activity together with increased biosynthesis resulted in -serine producers of with excellent production characteristics and maximal specific productivities of 1.45 mmol g h accumulating more than 50 g l-serine.

-Alanine, -aspartate, and -asparagine are non-essential amino acids that can be produced in microorganisms with various enzymes. -Alanine is produced by alanine transaminase (EC 2.6.1.2), alanine:oxo-acid aminotransferase (EC 2.6.1.12), β-alanine -pyruvate aminotransferase (EC 2.6.1.18), alanine-glyoxylate transaminase (EC 2.6.1.44), aspartate 1-decarboxylase (EC 4.1.1.11), aspartate 4-decarboxylase (EC 4.1.1.12), alanine racemase (EC 5.1.1.1), and alanine-tRNA ligase (EC 6.1.1.7). -Aspartate is produced by aspartate transaminase (EC 2.6.1.1), asparaginase (EC 3.5.1.1), aspartate ammonia-lyase (EC 4.3.1.1), aspartate racemase (EC 5.1.1.13), adenylosuccinate synthase (EC 6.3.4.4), argininosuccinate synthetase (EC 6.3.4.5), and asparagine synthase (EC 6.3.5.4). -Alanine, -aspartate, and -asparagine are converted to each other: -aspartate is converted to -alanine and -asparagine directly by -aspartate 4-decarboxylase and -aspartate:ammonia ligase (EC 6.3.1.1), respectively, whereas -asparagine is converted to -aspartate by -asparagine amidohydrolase (EC 3.5.1.1). Unusual amino acids such as -alanine and -aspartate are produced by alanine racemase (EC 5.1.1.1) and aspartate racemase (EC 5.1.1.13), respectively. The α-amino group of -alanine and -aspartate is transferred to -glutamate by -alanine:2-oxo-acid aminotransferase and -aspartate:2-oxoglutarate aminotransferase and is subsequently released as ammonia by glutamate dehydrogenase (EC 1.4.1.2). When these enzymes catalyze the reversed reactions, -alanine and -aspartate are synthesized via -glutamate from ammonia and pyruvate and from ammonia and oxaloacetate, respectively. -Alanine, -aspartate, -asparagine, -alanine, and -aspartate are useful as ingredients or starting materials for the industrial production of foods, cosmetics, medicines, and other products. For example, -alanine is widely used as a natural moisture balancer in various cosmetics to keep the skin moist. -Aspartate is one of the important raw materials for production of the artificial sweetener, aspartame. The -acyl derivatives of -alanine and -aspartate show antibotulinal activity in the presence of sodium nitrite, and are expected to be antibacterial agents (Paquet and Rayman 1987).