Metabolic engineering of Saccharomyces cerevisiae for the production of n-butanol

<p>Abstract</p> <p>Background</p> <p>Increasing energy costs and environmental concerns have motivated engineering microbes for the production of "second generation" biofuels that have better properties than ethanol.</p> <p>Results and conclusion</p> <p><it>Saccharomyces cerevisiae </it>was engineered with an n-butanol biosynthetic pathway, in which isozymes from a number of different organisms (<it>S. cerevisiae</it>, <it>Escherichia coli</it>, <it>Clostridium beijerinckii</it>, and <it>Ralstonia eutropha</it>) were substituted for the Clostridial enzymes and their effect on n-butanol production was compared. By choosing the appropriate isozymes, we were able to improve production of n-butanol ten-fold to 2.5 mg/L. The most productive strains harbored the <it>C. beijerinckii </it>3-hydroxybutyryl-CoA dehydrogenase, which uses NADH as a co-factor, rather than the <it>R. eutropha </it>isozyme, which uses NADPH, and the acetoacetyl-CoA transferase from <it>S. cerevisiae </it>or <it>E. coli </it>rather than that from <it>R. eutropha</it>. Surprisingly, expression of the genes encoding the butyryl-CoA dehydrogenase from <it>C. beijerinckii </it>(<it>bcd </it>and <it>etfAB</it>) did not improve butanol production significantly as previously reported in <it>E. coli</it>. Using metabolite analysis, we were able to determine which steps in the n-butanol biosynthetic pathway were the most problematic and ripe for future improvement.</p

計画1-2 和歌山県、奈良県の野生ニホンザル地域個体群とそれをめぐる社会的要因(VI 共同利用研究 2.研究成果)

Abstract Background As engineered biological systems become more complex, it is increasingly common to express multiple operons from different plasmids and inducible expression systems within a single host cell. Optimizing such systems often requires screening combinations of origins of replication, expression systems, and antibiotic markers. This procedure is hampered by a lack of quantitative data on how these components behave when more than one origin of replication or expression system are used simultaneously. Additionally, this process can be time consuming as it often requires the creation of new vectors or cloning into existing but disparate vectors. Results Here, we report the development and characterization of a library of expression vectors compatible with the BglBrick standard (BBF RFC 21). We have designed and constructed 96 BglBrick-compatible plasmids with a combination of replication origins, antibiotic resistance genes, and inducible promoters. These plasmids were characterized over a range of inducer concentrations, in the presence of non-cognate inducer molecules, and with several growth media, and their characteristics were documented in a standard format datasheet. A three plasmid system was used to investigate the impact of multiple origins of replication on plasmid copy number. Conclusions The standardized collection of vectors presented here allows the user to rapidly construct and test the expression of genes with various combinations of promoter strength, inducible expression system, copy number, and antibiotic resistance. The quantitative datasheets created for these vectors will increase the predictability of gene expression, especially when multiple plasmids and inducers are utilized

Evaluation of therapeutic potential of VB-001, a leave-on formulation, for the treatment of moderate adherent dandruff

Background: Dandruff is a common scalp condition characterized by excessive scaling and itch. Aberrant colonization of the scalp by commensal Malassezia spp. is a major contributor in the multifactorial etiology of dandruff. Literature based understanding of Malassezia linked pathophysiology of dandruff allowed us to comprehend a strategy to potentiate the efficacy of a known antifungal agent used in dandruff therapy. The aim of this study was to determine the efficacy and skin safety of VB-001 antidandruff leave-on formulation in comparison with marketed antidandruff ZPTO shampoo in patients with moderate adherent dandruff of the scalp. Methods: Healthy males or females aged ≥ 15 years and ≤ 65 with a clinical diagnosis of moderate adherent dandruff of the scalp were recruited for the study to monitor the effects of topical VB-001 versus those of marketed antidandruff ZPTO shampoo. Results: 168 subjects were randomized to the treatment (VB-001, n = 84) and control (ZPTO shampoo, n = 84) groups. The efficacy of each product was evaluated by comparing proportion of subjects who have shown reduction in flaking by ASFS (adherent scalp flaking score) and pruritus by IGA (investigator global assessment) score. VB-001 imparted consistently better reduction in ASFS and enabled early reduction of pruritus in comparison to marketed ZPTO shampoo. Conclusion: VB-001, a leave-on formulation with ingredients chosen to selectively disturb the Malassezia niche on dandruff scalp by denying extra nutritional benefits to the microbe, provides unique advantages over existing best in class ZPTO shampoo therapy. It has the potential to emerge as an attractive novel treatment for moderate adherent dandruff. Trial registration CTRI Registration number: CTRI/2013/01/003283. Registered on: 02/01/201

BglBrick vectors and datasheets: A synthetic biology platform for gene expression.

BackgroundAs engineered biological systems become more complex, it is increasingly common to express multiple operons from different plasmids and inducible expression systems within a single host cell. Optimizing such systems often requires screening combinations of origins of replication, expression systems, and antibiotic markers. This procedure is hampered by a lack of quantitative data on how these components behave when more than one origin of replication or expression system are used simultaneously. Additionally, this process can be time consuming as it often requires the creation of new vectors or cloning into existing but disparate vectors.ResultsHere, we report the development and characterization of a library of expression vectors compatible with the BglBrick standard (BBF RFC 21). We have designed and constructed 96 BglBrick-compatible plasmids with a combination of replication origins, antibiotic resistance genes, and inducible promoters. These plasmids were characterized over a range of inducer concentrations, in the presence of non-cognate inducer molecules, and with several growth media, and their characteristics were documented in a standard format datasheet. A three plasmid system was used to investigate the impact of multiple origins of replication on plasmid copy number.ConclusionsThe standardized collection of vectors presented here allows the user to rapidly construct and test the expression of genes with various combinations of promoter strength, inducible expression system, copy number, and antibiotic resistance. The quantitative datasheets created for these vectors will increase the predictability of gene expression, especially when multiple plasmids and inducers are utilized