To provide methods for effectively producing 1,4-butanediol (BOD) and chemical precursors thereof in commercial quantities.SOLUTION: Disclosed herein is a non-naturally occurring microorganism comprising at least one exogenous nucleic acid encoding an enzyme in BOD production pathway to express the exogenous enzyme with a disrupted gene encoding an aerobic respiratory control regulatory system. The exogenous enzymes are succinyl-CoA synthetase, alpha-ketoglutarate decarboxylase, succinate semialdehyde dehydrogenase and 4-hydroxybutyrate dehydrogenase and optionally 4-hydroxybutyryl-CoA/acetyl-CoA transferase, butyrate kinase and phosphotransbutyrase, 4-hydroxybutyryl-CoA reductase, 4-hydroxybutanal reductase, pyruvate dehydrogenase, NADH-insensitive citrate synthetase and phosphoenolpyruvate carboxykinase.SELECTED DRAWING: Figure 46

Genomatica has established an integrated computational/experimental metabolic engineering platform to design, create, and optimize novel high performance organisms and bioprocesses. Here we present our platform and its use to develop E. coli strains for production of the industrial chemical 1,4-butanediol (BDO) from sugars. A series of examples are given to demonstrate how a rational approach to strain engineering, including carefully designed diagnostic experiments, provided critical insights about pathway bottlenecks, byproducts, expression balancing, and commercial robustness, leading to a superior BDO production strain and process.

L'invention concerne des organismes microbiens d'origine non naturelle qui presentent une voie de production de 4-hydroxybutyrate, 1,4-butanediol ou d'un autre produit et qui sont aptes a produire du 4-hydroxybutyrate, 1,4-butanediol, ou un autre produit, l'organisme microbien comportant une ou plusieurs modifications genetiques. L'invention concerne egalement des procedes de production de 4-hydroxybutyrate, 1,4-butanediol ou d'un autre produit ou de produits associes a l'aide des organismes microbiens.

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.

L'invention concerne des organismes microbiens d'origine non naturelle comprenant une voie 1,4- butanediol (BDO), 4-hydroxybutyryl-CoA, 4-hydroxybutanal ou putrescine comprenant au moins un acide nucleique exogene codant pour une enzyme de la voie BDO, 4-hydroxybutyryl-CoA, 4-hydroxybutanal ou putrescine en quantite suffisante pour produire BDO, 4-hydroxybutyryl-CoA, 4-hydroxybutanal ou putrescine et optimiser encore l'expression de BDO. L'invention porte egalement sur des methodes d'utilisation desdits organismes microbiens pour produire BDO, 4-hydroxybutyryl-CoA, 4-hydroxybutanal ou putrescine.

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.

Cette invention concerne des organismes microbiens non naturels comprenant une voie du 1,4-butanediol (BDO) et comprenant au moins un acide nucleique exogene codant pour une enzyme de la voie du BDO exprimee en une quantite suffisante pour produire du BDO et, en outre, optimise pour l'expression du BDO. Cette invention concerne, en plus, des procedes d'utilisation de ces organismes microbiens pour produire du BDO.

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.

The overall process of antimicrobial drug discovery and development seems simple, to cure infectious disease by identifying suitable antibiotic drugs. However, this goal has been difficult to fulfill in recent years. Despite the promise of the high-throughput innovations sparked by the genomics revolution, discovery, and development of new antibiotics has lagged in recent years exacerbating the already serious problem of evolution of antibiotic resistance. Therefore, both new antimicrobials are desperately needed as are improvements to speed up or improve nearly all steps in the process of discovering novel antibiotics and bringing these to clinical use. Another product of the genomic revolution is the modeling of metabolism using computational methodologies. Genomic-scale networks of metabolic reactions based on stoichiometry, thermodynamics and other physico-chemical constraints that emulate microbial metabolism have been developed into valuable research tools in metabolic engineering and other fields. This constraint-based modeling is predictive in identifying critical reactions, metabolites, and genes in metabolism. This is extremely useful in determining and rationalizing cellular metabolic requirements. In turn, these methods can be used to predict potential metabolic targets for antimicrobial research especially if used to increase the confidence in prioritization of metabolic targets. The many different capacities of constraint-based modeling also enable prediction of cellular response to specific inhibitors such as antibiotics and this may, ultimately find a role in drug discovery and development. Herein, we describe the principles of metabolic modeling and how they might initially be applied to antimicrobial research.