Trond Erling Ellingsen

In Vivo Analysis of the Regulatory Genes in the Nystatin Biosynthetic Gene Cluster of Streptomyces noursei ATCC 11455 Reveals Their Differential Control Over Antibiotic Biosynthesis

Six putative regulatory genes are located at the flank of the nystatin biosynthetic gene cluster in Streptomyces noursei ATCC 11455. Gene inactivation and complementation experiments revealed that nysRI, nysRII, nysRIII, and nysRIV are necessary for efficient nystatin production, whereas no significant roles could be demonstrated for the other two regulatory genes. To determine the in vivo targets for the NysR regulators, chromosomal integration vectors with the xylE reporter gene under the control of seven putative promoter regions upstream of the nystatin structural and regulatory genes were constructed. Expression analyses of the resulting vectors in the S. noursei wild-type strain and regulatory mutants revealed that the four regulators differentially affect certain promoters. According to these analyses, genes responsible for initiation of nystatin biosynthesis and antibiotic transport were the major targets for regulation. Data from cross-complementation experiments showed that nysR genes could in some cases substitute for each other, suggesting a functional hierarchy of the regulators and implying a cascade-like mechanism of regulation of nystatin biosynthesis.

Analysis and Manipulation of Aspartate Pathway Genes for l-Lysine Overproduction from Methanol by Bacillus methanolicus

ABSTRACT We investigated the regulation and roles of six aspartate pathway genes in l-lysine overproduction in Bacillus methanolicus: dapG, encoding aspartokinase I (AKI); lysC, encoding AKII; yclM, encoding AKIII; asd, encoding aspartate semialdehyde dehydrogenase; dapA, encoding dihydrodipicolinate synthase; and lysA, encoding meso-diaminopimelate decarboxylase. Analysis of the wild-type strain revealed that in vivo lysC transcription was repressed 5-fold by l-lysine and induced 2-fold by dl-methionine added to the growth medium. Surprisingly, yclM transcription was repressed 5-fold by dl-methionine, while the dapG, asd, dapA, and lysA genes were not significantly repressed by any of the aspartate pathway amino acids. We show that the l-lysine-overproducing classical B. methanolicus mutant NOA2#13A52-8A66 has—in addition to a hom-1 mutation—chromosomal mutations in the dapG coding region and in the lysA promoter region. No mutations were found in its dapA, lysC, asd, and yclM genes. The mutant dapG gene product had abolished feedback inhibition by meso-diaminopimelate in vitro, and the lysA mutation was accompanied by an elevated (6-fold) lysA transcription level in vivo. Moreover, yclM transcription was increased 16-fold in mutant strain NOA2#13A52-8A66 compared to the wild-type strain. Overexpression of wild-type and mutant aspartate pathway genes demonstrated that all six genes are important for l-lysine overproduction as tested in shake flasks, and the effects were dependent on the genetic background tested. Coupled overexpression of up to three genes resulted in additive (above 80-fold) increased l-lysine production levels.

Lysine Industrial Uses and Production

l-Lysine belongs to the essential amino acids that cannot be synthesized by higher animals and humans. It is an essential ingredient for growth of animals and must therefore be available in animal feed to meet the nutritional requirements and to ensure a balanced diet. l-Lysine is widely used as feed supplement for poultry, swine, and other livestock and is also used in pharmaceuticals, dietary supplements, and cosmetics. Therefore, l-lysine is a biotechnological product of considerable economic importance, and the worldwide production was recently estimated to be above 1 million tons per year. Most bacteria can synthesize l-lysine and industrial production of l-lysine is almost entirely by microbial fermentation. The most important industrial l-lysine producer is the bacterium Corynebacterium glutamicum and several decades of extensive research has resulted in a detailed insight into its physiology, biochemistry, and genetics. This knowledge has been used to generate efficient l-lysine-overproducing strains by systems-level metabolic engineering. Along with this, fermentation technology and downstream processes are constantly being optimized. The carbon sources for C. glutamicum fermentations are sugars from agricultural crops. In recent years, alternatives to sugars, such as lignocelluloses and methanol, have gained increased interest. Alternative approaches including extending the substrate range by genetic engineering, as well as adopting methylotrophic bacteria for l-lysine production, will be discussed.