Corynebacterium glutamicum has played a principal role in the progress of the amino acid fermentation industry. The complete genome sequence of the representative wild-type strain of C. glutamicum, ATCC 13032, has been determined and analyzed to improve our understanding of the molecular biology and physiology of this organism, and to advance the development of more efficient production strains. Genome annotation has helped in elucidation of the gene repertoire defining a desired pathway, which is accelerating pathway engineering. Post genome technologies such as DNA arrays and proteomics are currently undergoing rapid development in C. glutamicum. Such progress has already exposed new regulatory networks and functions that had so far been unidentified in this microbe. The next goal of these studies is to integrate the fruits of genomics into strain development technology. A novel methodology that merges genomics with classical strain improvement has been developed and applied for the reconstruction of classically derived production strains. How can traditional fermentation benefit from the C. glutamicum genomic data? The path from genomics to biotechnological processes is presented.

The Corynebacterium glutamicum genome: features and impacts on biotechnological processes

With the exploitation of new uses and the growing markets of amino acids, amino acid production technology has made large progress during the latter half of the 20th century. Fermentation technology has played crucial roles in this progress, and currently the fermented amino acids represent chief products of biotechnology in both volume and value. This area is highly competitive in the world market and process economics are of primary importance. For cost-effective production, many technologies have been developed to establish high-productive fermentation and recovery processes. The producer organisms used in large-scale, well-established processes have been developed to a high level of production efficiency. The tools of genetic engineering of amino acid-producing organisms have been well developed and are now being applied for enlargement of biosynthetic and transport capacity, which is beginning to have a great impact on the amino acid industry. Furthermore, the rapid strides in genome analysis are bound to revolutionize the strain improvement methodology.

Amino acid production processes.

The aromatic amino acids, L-tryptophan, L-phenylalanine, and L-tyrosine, can be manufactured by bacterial fermentation. Until recently, production efficiency of classical aromatic amino-acid-producing mutants had not yet reached a high level enough to make the fermentation method the most economic. With the introduction of recombinant DNA technology, it has become possible to apply more rational approaches to strain improvement. Many recent activities in this metabolic engineering have led to several effective approaches, which include modification of terminal pathways leading to removal of bottleneck or metabolic conversion, engineering of central carbon metabolism leading to increased supply of precursors, and transport engineering leading to reduced intracellular pool of the aromatic amino acids. In this review, advances in metabolic engineering for the production of the aromatic amino acids and useful aromatic intermediates are described with particular emphasis on two representative producer organisms, Corynebacterium glutamicum and Escherichia coli.

Towards bacterial strains overproducing L-tryptophan and other aromatics by metabolic engineering

Genetic and physiological approaches for the production of amino acids

Amino Acids – Technical Production and Use

Introduction of plasmid pKW99, which coexpresses the deregulated 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and tryptophan-biosynthetic enzymes, into tryptophan-producing Corynebacterium glutamicum KY10894 resulted in a marked increase (54%) in yield of tryptophan production (43 g/liter), but incurred two problems. One was a decline in sugar consumption at the late stage of fermentation, and the other the loss of the plasmid in the absence of selective pressure. The retarded sugar assimilation was found to be attributed to the death of cells that arose from the detrimental action of indole, the last intermediate in the tryptophan pathway, accumulated as a by-product. A chain of these events simultaneously disappeared when serine, the other substrate of the final reaction by tryptophan synthase, was added. These results indicated that a limiting supply of serine was the cause of the decline in the sugar consumption. Thus, to increase carbon flux into serine, the gene for 3-phosphoglycerate dehydrogenase (PGD), the first enzyme in the serine pathway, was cloned from wild-type C. glutamicum ATCC 31833 and joined onto pKW99 to generate pKW9901. Strain KY10894 transformed with pKW9901 favorably consumed sugar through fermentation with accumulating little indole. Furthermore, on the basis of the observation that serine in the medium was consumed rapidly by the recombinant cells, we developed a unique plasmid stabilization system composed of KY9218 (a PGD-deficient serine-requiring strain of KY10894) and pKW9901: In its combination, cells lacking the plasmid should not proliferate in the fermentation medium which does not contain serine. Even if selective pressure was not applied, the modified strain KY9218 with pKW9901 stably maintained the plasmid during fermentation and produced 50 g/liter of tryptophan in a 61% increased yield relative to strain KY10894.

Fermentative production of tryptophan by a stable recombinant strain of Corynebacterium glutamicum with a modified serine-biosynthetic pathway.

Described are coryneform microorganisms capable of assimilating lactose which carry a recombinant DNA capable of conferring the ability to assimilate lactose on coryneform microorganisms, and a process for producing L-­amino acids which comprises culturing said coryneform microorganism capable of assimilating lactose in a culture medium containing lactose to form an amino acid, and recovering said amino acid accumulated in the culture broth. Further, the invention relates to a method for preparing recombinant plasmids containing a DNA fragment essential to the expression of a gene in coryneform microorganisms.

Novel microorganisms capable of assimilating lactose

The present invention provides a process for producing substances such as L-tryptophan and L-threonine, which comprises transforming a serine-requiring microorganism belonging to the genus Corynebacterium or Brevibacterium by incorporation of a recombinant plasmid containing a gene which complements the serine-requirement of the host and a gene which relates to the biosynthesis of a desired substance; culturing the obtained transformant in a culture medium; allowing the substance produced by the transformant to accumulate in the culture; and recovering the substance from the culture.

Process for producing L-tryptophan and L-threonine

L-tryptophan is produced by constructing a recombinant DNA composed of a vector DNA and DNA fragments bearing all of genetic information relating to the synthesis of DS, AS, PRT, PRAI, InGPS, TS and PGDH, introducing the recombinant DNA into a microorganism belonging to the genus Corynebacterium or Brevibacterium, culturing the microorganism in a medium, and recovering L-tryptophan accumulated in the culture.

Recombinant coryneform bacterium for producing L-tryptophan

PURPOSE: To improve productivity by culturing a bacterium belonging to the genus Corynebacterium or brevibacterium containing specific DNA in a medium. CONSTITUTION: A DNA fragment (A) containing the whole of genetic information taking part in synthesis of 3-deoxy-D-arabino-hepturonate-7-phosphate synthase, anthranilic acid synthase, anthranilic acid phosphoribosyl transferase, N-5'-phosphoribosylanthranilic acid isomerase, indole-3-glycerol phosphate synthase, tryptophan synthase and 3-phosphoglycerate dehydrogenase is obtained by induction from a bacterium belonging to the genus Corynebacterium or Brevibacterium. Then the component A is bonded to vector DNA (e.g. plasmid pCG1) (B) to give a recombinant DNA (C). Then a bacterium belonging to the genus Corynebacterium is transduced with the component C to give a transformant, which is cultured in a medium containing glucose, etc., at 20-40°C for 1-5 days and L-tryptophan is recovered from the culture mixture. COPYRIGHT: (C)1991,JPO&Japio

Keiko Nakanishi

Papers

Fermentative production of tryptophan by a stable recombinant strain of Corynebacterium glutamicum with a modified serine-biosynthetic pathway.

Novel microorganisms capable of assimilating lactose

Process for producing L-tryptophan and L-threonine

Recombinant coryneform bacterium for producing L-tryptophan

Production of l-tryptophan