1,4-Butanediol (BDO) is an important commodity chemical used to manufacture over 2.5 million tons annually of valuable polymers, and it is currently produced exclusively through feedstocks derived from oil and natural gas. Herein we report what are to our knowledge the first direct biocatalytic routes to BDO from renewable carbohydrate feedstocks, leading to a strain of Escherichia coli capable of producing 18 g l(-1) of this highly reduced, non-natural chemical. A pathway-identification algorithm elucidated multiple pathways for the biosynthesis of BDO from common metabolic intermediates. Guided by a genome-scale metabolic model, we engineered the E. coli host to enhance anaerobic operation of the oxidative tricarboxylic acid cycle, thereby generating reducing power to drive the BDO pathway. The organism produced BDO from glucose, xylose, sucrose and biomass-derived mixed sugar streams. This work demonstrates a systems-based metabolic engineering approach to strain design and development that can enable new bioprocesses for commodity chemicals that are not naturally produced by living cells.
The widespread emergence of antibiotic-resistant bacteria and a lack of new pharmaceutical development have catalyzed a need for new and innovative approaches for antibiotic drug discovery. One bottleneck in antibiotic discovery is the lack of a rapid and comprehensive method to identify compound mode of action (MOA). Since a hallmark of antibiotic action is as an inhibitor of essential cellular targets and processes, we identify a set of 308 essential genes in the clinically important pathogen Staphylococcus aureus. A total of 446 strains differentially expressing these genes were constructed in a comprehensive platform of sensitized and resistant strains. A subset of strains allows either target underexpression or target overexpression by heterologous promoter replacements with a suite of tetracycline-regulatable promoters. A further subset of 236 antisense RNA-expressing clones allows knockdown expression of cognate targets. Knockdown expression confers selective antibiotic hypersensitivity, while target overexpression confers resistance. The antisense strains were configured into a TargetArray in which pools of sensitized strains were challenged in fitness tests. A rapid detection method measures strain responses toward antibiotics. The TargetArray antibiotic fitness test results show mechanistically informative biological fingerprints that allow MOA elucidation.
Mitochondrial-nuclear communication is taking on increased importance in models of oxygen sensing, oxidative stress, aging, and disease. The deletion of the mitochondrial genome (mtDNA) and, hence, the ability to respire, affects expression of several nuclear genes through at least two different mitochondrial-nuclear communication pathways. One of the pathways, retrograde regulation, is activated by a reduction in respiration, while another, intergenomic signaling, is unaffected by respiration but requires mtDNA. Using DNA microarrays, we identify here a set of nuclear genes in Saccharomyces cerevisiae that are targets of intergenomic signaling. These nuclear genes are down-regulated in rho degrees cells that lack mtDNA but not in nuclear pet mutant rho(+)cells that possess mtDNA but lack respiration. Many of these nuclear genes encode mitochondrial proteins, implying that intergenomic signaling functions in coordinating mitochondrial and nuclear gene expression. In addition, analyses of deletion and linker scanning mutations in the promoter of the COX6 gene, a nuclear gene affected by intergenomic signaling, suggest an involvement of Abf1p transcription factor in intergenomic signaling. Together, these findings indicate that intergenomic signaling is distinct from retrograde regulation both in the nuclear genes that it regulates and in the way in which it affects their expression.
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