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.

SummaryTo address the need for new approaches to antibiotic drug development, we have identified a large number of essential genes for the bacterial pathogen, Staphylococcus aureus, using a rapid shotgun antisense RNA method. Staphylococcus aureus chromosomal DNA fragments were cloned into a xylose-inducible expression plasmid and transformed into S. aureus. Homology comparisons between 658 S. aureus genes identified in this particular antisense screen and the Mycoplasma genitalium genome, which contains 517 genes in total, yielded 168 conserved genes, many of which appear to be essential in M. genitalium and other bacteria. Examples are presented in which expression of an antisense RNA specifically reduces its cognate mRNA. A cell-based, drug-screening assay is also described, wherein expression of an antisense RNA confers specific sensitivity to compounds targeting that gene product. This approach enables facile assay development for high throughput screening for any essential gene, independent of its biochemical function, thereby greatly facilitating the search for new antibiotics.

To explore the process of lipoprotein assembly, plasmids encoding truncated forms of apolipoprotein B (apoB) were transfected into Chinese hamster ovary (CHO) fibroblasts. (One, encoding apoB53, the N-terminal 53% of apoB100, can direct the assembly and secretion of lipoproteins when expressed in hepatoma cells, while the other, encoding the shorter apoB15, does not direct lipoprotein assembly.) Expression of apoB15 in CHO cells resulted in the accumulation of apoB15 protein in both medium and cells. In contrast, apoB was not detectable in medium or within CHO cells transfected with the plasmid encoding apoB53, despite the expression of apoB53 mRNA. ApoB53 did accumulate within transfected cells incubated with the thiol protease inhibitor N-acetylleucylleucylnorleucinal (ALLN), suggesting that it is synthesized but completely degraded in the absence of the inhibitor. ApoB53 was not secreted despite its presence within ALLN-treated cells. Essentially all the apoB53 that accumulated in microsomes from ALLN-treated cells was associated with the membrane and was susceptible to degradation by exogenous trypsin, indicating exposure on the cytoplasmic face of the membrane. Thus, translocation of apoB53 across the endoplasmic reticulum membrane is blocked. However, the apoB53 bound to concanavalin A, suggesting that it is glycosylated and therefore partly exposed to the lumen as well. ApoB requires a unique process, not expressed in CHO fibroblasts, for its complete translocation and entrance into the secretory pathway. This process might account for the inability of abetalipoproteinemic patients to secrete apoB.Apolipoprotein B (apoB) is a large amphipathic protein responsible for the assembly of very low density lipoprotein and chylomicrons by the liver and intestine (1). Abetalipoproteinemia is a recessive disease characterized by the absence of plasma apoB (2), although the disease is not linked to the gene encoding apoB (3). Livers of patients with abetalipoproteinemia have apoB mRNA of normal size and elevated abundance (4). ApoB is synthesized (5, 6); however, almost no apoB is secreted (2). Triglyceride-rich lipoproteins are not demonstrable in any organelle of the secretory pathway (2, 6), suggesting that the block in secretion occurs prior to translocation or assembly into lipoproteins. The inability to secrete apoB is not caused by a general impairment of protein secretion, since the plasma concentrations of other liverderived proteins are not altered in abetalipoproteinemics (2). Thus, apoB requires a unique process, absent in homozygous abetalipoproteinemia, for its secretion.When expressed in hepatoma cells, apoB53 (the N-terminal 53% of apoB100, encompassing the sequence of the naturally occurring apoB48) assembles lipoproteins and is secreted as a lipoprotein complex (7). In marked contrast, when apoB53 is expressed in nonhepatic COS cells, it is not secreted (7). While the inability of COS cells to secrete apoB53 could be an artifact of overexpression, it might instead reflect tissue specific...

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.

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.