Metabolic engineering of Escherichia coli for direct production of 1,4-butanediol.

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.

Defined salt formulations for the growth of Salinispora tropica strain NPS21184 and the production of salinosporamide A (NPI-0052) and related analogs.

Salinosporamide A (NPI-0052) is currently produced by a marine actinomycete, Salinispora tropica, via a saline fermentation process using a non-defined, commercially available synthetic sea salt, Instant Ocean. In order to control the consistency of the production of NPI-0052 and related analogs, two chemically defined salt formulations were developed to replace Instant Ocean. A chemically defined sodium-chloride-based salt formulation with similar sodium and chloride contents as in Instant Ocean was found to support higher production of NPI-0052 and a better metabolite production profile for downstream processing than Instant Ocean. A chemically defined sodium-sulfate-based salt formulation with low chloride concentration at 17 mM was found to support a similar NPI-0052 and metabolite production profile as Instant Ocean. The sodium-sulfate-based formulation is a robust formulation for large-scale production process due to its reduced corrosiveness in fermentation as compared with the saline fermentation utilizing Instant Ocean or the sodium-chloride-based salt formulation. The production of NPI-0052 in both chemically defined salt formulations was successfully scaled-up to a 42-l fermentor, indicating that these salt formulations can be used for large-scale manufacturing process.

Seawater requirement for the production of lipoxazolidinones by marine actinomycete strain NPS8920.

A novel marine actinomycete strain NPS8920 produces a new class of 4-oxazolidinone antibiotics lipoxazolidinone A, B and C. Lipoxazolidinone A possesses good potency (1-2 microg/mL) against drug-resistant pathogens methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE). Strain NPS8920 exhibits different morphologies in both agar and submerged cultures. The ability of strain NPS8920 to sporulate on saline-based agar media but not on deionized water-based agar medium supported that strain NPS8920 is a marine actinomycete. While strain NPS8920 does not require seawater for growth, the production of lipoxazolidinones by strain NPS8920 can only be detected in the seawater-based media. The optimal production of lipoxazolidinones was observed in the natural seawater-based medium. Strain NPS8920 produced 10-20% of lipoxazolidinones in the synthetic sea salt Instant Ocean-based medium and no production in the sodium chloride-based and deionized water-based media.

Lipoxazolidinones A, B, and C: antibacterial 4-oxazolidinones from a marine actinomycete isolated from a Guam marine sediment.

Marine actinomycete strain NPS008920, a member of the new genus Marinispora, was isolated from a sediment sample collected in Cocos Lagoon, Guam. In natural sea water containing media, the strain produced a series of novel 2-alkylidene-5-alkyl-4-oxazolidinones, lipoxazolidinone A (1), B (2), and C (3). Compounds 1- 3 showed broad spectrum antimicrobial activity similar to that of the commercial antibiotic linezolid (Zyvox), a 2-oxazolidinone. Hydrolysis of the amide bond of the 4-oxazolidinone ring of 1 resulted in loss of antibacterial activity. The 2-alkylidene-4-oxazolidinone represents a new antibiotic pharmacophore and is unprecedented in nature.

Salinosporamides D-J from the marine actinomycete Salinispora tropica, bromosalinosporamide, and thioester derivatives are potent inhibitors of the 20S proteasome.

Salinosporamide A (NPI-0052; 3), a highly potent inhibitor of the 20S proteasome, is currently in phase I clinical trials for the treatment of cancer. During the course of purifying multigram quantities of 3 from Salinispora tropica fermentation extracts, several new salinosporamides were isolated and characterized, most of which represent modifications to the chloroethyl substituent at C-2. Specifically, 3 was isolated along with the known compound salinosporamide B (4), the previously undescribed methyl congener salinosporamide D (7), and C-2 epimers of 3 and 7 (salinosporamides F (9) and G (10), respectively). Salinosporamide I (13), in which the methyl group at the ring junction is replaced with an ethyl group, and the C-5 deshydroxyl analogue salinosporamide J (14), were also identified. Replacement of synthetic sea salt with sodium bromide in the fermentation media produced bromosalinosporamide (12), 4, and its C-2 epimer (11, salinosporamide H). In addition to these eight new salinosporamides, several thioester derivatives were generated semisynthetically. IC50 values for cytotoxicity against human multiple myeloma cell line RPMI 8226 and inhibition of the chymotrypsin-like (CT-L) activity of purified rabbit 20S proteasomes were determined for all compounds. The results indicate that thioesters may directly inhibit the proteasome, albeit with reduced potency compared to their beta-lactone counterparts.

Glaciapyrroles A, B, and C, pyrrolosesquiterpenes from a Streptomyces sp. isolated from an Alaskan marine sediment.

A Streptomyces sp. (NPS008187) isolated from a marine sediment collected in Alaska was found to produce three new pyrrolosesquiterpenes, glyciapyrroles A (1), B (2), and C (3), along with the known diketopiperazines cyclo(leucyl-prolyl) (4), cyclo(isoleucyl-prolyl) (5), and cyclo(phenylalanyl-prolyl) (6). The structures of 1, 2, and 3 were established using spectroscopic methods.

Lajollamycin, a nitro-tetraene spiro-beta-lactone-gamma-lactam antibiotic from the marine actinomycete Streptomyces nodosus.

A strain of Streptomyces nodosus (NPS007994) isolated from a marine sediment collected in Scripps Canyon, La Jolla, California, was found to produce lajollamycin (1), a nitro-tetraene spiro-beta-lactone-gamma-lactam antibiotic. The structure was established by complete analysis of spectroscopic data and comparison with known antibiotics oxazolomycin (2), 16-methyloxazolomycin (3), and triedimycin B (4). Lajollamycin (1) showed antimicrobial activity against both drug-sensitive and -resistant Gram-positive bacteria and inhibited the growth of B16-F10 tumor cells in vitro.

Aureoverticillactam, a novel 22-atom macrocyclic lactam from the marine actinomycete Streptomyces aureoverticillatus.

During the course of our screening program designed to discover novel anticancer and anti-infective agents from marine microorganisms, a strain of Streptomyces aureoverticillatus (NPS001583) isolated from a marine sediment was found to produce a novel macrocyclic lactam with cytotoxicity against various tumor cell lines. Using extensive MS, UV, and NMR spectral analyses, the structure has been established as compound 1, aureoverticillactam, a 22-atom macrocyclic lactam incorporating both triene and tetraene conjugated olefins.