Metabolic Engineering of Cofactor F420 Production in Mycobacterium smegmatis

Cofactor F420 is a unique electron carrier in a number of microorganisms including Archaea and Mycobacteria. It has been shown that F420 has a direct and important role in archaeal energy metabolism whereas the role of F420 in mycobacterial metabolism has only begun to be uncovered in the last few years. It has been suggested that cofactor F420 has a role in the pathogenesis of M. tuberculosis, the causative agent of tuberculosis. In the absence of a commercial source for F420, M. smegmatis has previously been used to provide this cofactor for studies of the F420-dependent proteins from mycobacterial species. Three proteins have been shown to be involved in the F420 biosynthesis in Mycobacteria and three other proteins have been demonstrated to be involved in F420 metabolism. Here we report the over-expression of all of these proteins in M. smegmatis and testing of their importance for F420 production. The results indicate that co–expression of the F420 biosynthetic proteins can give rise to a much higher F420 production level. This was achieved by designing and preparing a new T7 promoter–based co-expression shuttle vector. A combination of co–expression of the F420 biosynthetic proteins and fine-tuning of the culture media has enabled us to achieve F420 production levels of up to 10 times higher compared with the wild type M. smegmatis strain. The high levels of the F420 produced in this study provide a suitable source of this cofactor for studies of F420-dependent proteins from other microorganisms and for possible biotechnological applications

Supplementary data on the characterization and safety evaluation of HPPD W336, a modified 4-hydroxyphenylpyruvate dioxygenase protein, which confers herbicide tolerance, and on the compositional assessment of field grown MST-FGØ72-2 soybean expressing HPPD W336

Supplementary data are provided which are supportive to the research article entitled “Characterization and safety evaluation of HPPD W336, a modified 4-hydroxyphenylpyruvate dioxygenase protein, and the impact of its expression on plant metabolism in herbicide-tolerant MST-FGØ72-2 soybean” (Dreesen et al., 2018) [1]. The conducted supplementary analyses include the characterization of additional Escherichia coli-produced HPPD W336 protein batches used as a surrogate in HPPD W336 safety studies, the assessment of potential glycosylation and monitoring of stability in simulated intestinal fluid and during heating of the HPPD W336 protein. Furthermore, data are provided on conducted field trials and subsequent compositional analysis in MST-FGØ72-2 soybean grain of compounds related to the tyrosine degradation pathway and the metabolism of homogentisate

Stories For Children, Histories of Childhood. Volume II

The place of the child and childhood in our culture and his/her legal status is a subject which touches a sensitive nerve and triggers passionate responses just as much at the start of the 21st century as it did two hundred years ago. Curious then to note that the academic study of childhood had long been neglected by social historians while its literature, with a few notable exceptions, had too often been relegated to a minor category when not simply dismissed as "pulp fiction". Over the last two decades or so pioneering research has begun to redress this balance and paved the way towards a reappraisal of the child and childhood as a valid field of study. At the same time, by highlighting the areas which still require exploration, it has underlined the distance we still have to cover in order to achieve a balanced integration of both the child and childhood into the social and cultural "story" of our past. It is hoped that the papers published here will, in their own modest way, contribute to this ongoing process of replacing the child inside a culture which proudly claims to have created the golden age of childhood

Genetic Stability, Inheritance Patterns and Expression Stability in Biotech Crops

Demonstration of the stability of traits newly introduced into a plant genome via genetic engineering approaches comprise a significant portion of the safety assessment that these products undergo prior to receiving the requisite regulatory approvals enabling commercial authorization. Different regions of the world have different regulatory requirements and many ask similar questions from multiple and overlapping perspectives. The entire central dogma, that is stability at the DNA level, mRNA level and protein level, is assessed for each product, although only a few regulatory authorities request data at the mRNA level. In this article, we present inheritance data obtained during the safety assessment of biotech products representing specific transgenic events in several crop species including Brassica napus (canola); canola quality Brassica juncea (yellow seeded canola); Glycine max (soybean), and Gossypium hirsutum (cotton) in which different traits have been introduced. The data presented confirm that all events examined were nuclear insertions that resulted in typical Mendelian Inheritance patterns and that the proteins are expressed similarly across multiple generations regardless of whether they were from backcrossed or outcrossed generations. These results demonstrate that newly inserted genes are transmitted to their progeny in a stable manner similar to that of endogenous genes. Further, the findings demonstrate that assessments of multigenerational stability have very limited value to a safety assessment.</jats:p