In spite of its great promise, metabolomics has proven difficult to execute in an untargeted and generalizable manner. Liquid chromatography–mass spectrometry (LC–MS) has made it possible to gather data on thousands of cellular metabolites. However, matching metabolites to their spectral features continues to be a bottleneck, meaning that much of the collected information remains uninterpreted and that new metabolites are seldom discovered in untargeted studies. These challenges require new approaches that consider compounds beyond those available in curated biochemistry databases. Here we present Metabolic In silico Network Expansions (MINEs), an extension of known metabolite databases to include molecules that have not been observed, but are likely to occur based on known metabolites and common biochemical reactions. We utilize an algorithm called the Biochemical Network Integrated Computational Explorer (BNICE) and expert-curated reaction rules based on the Enzyme Commission classification system to propose the novel chemical structures and reactions that comprise MINE databases. Starting from the Kyoto Encyclopedia of Genes and Genomes (KEGG) COMPOUND database, the MINE contains over 571,000 compounds, of which 93% are not present in the PubChem database. However, these MINE compounds have on average higher structural similarity to natural products than compounds from KEGG or PubChem. MINE databases were able to propose annotations for 98.6% of a set of 667 MassBank spectra, 14% more than KEGG alone and equivalent to PubChem while returning far fewer candidates per spectra than PubChem (46 vs. 1715 median candidates). Application of MINEs to LC–MS accurate mass data enabled the identity of an unknown peak to be confidently predicted. MINE databases are freely accessible for non-commercial use via user-friendly web-tools at http://minedatabase.mcs.anl.gov and developer-friendly APIs. MINEs improve metabolomics peak identification as compared to general chemical databases whose results include irrelevant synthetic compounds. Furthermore, MINEs complement and expand on previous in silico generated compound databases that focus on human metabolism. We are actively developing the database; future versions of this resource will incorporate transformation rules for spontaneous chemical reactions and more advanced filtering and prioritization of candidate structures.

Like other forms of engineering, metabolic engineering requires knowledge of the components (the ‘parts list’) of the target system. Lack of such knowledge impairs both rational engineering design and diagnosis of the reasons for failures; it also poses problems for the related field of metabolic reconstruction, which uses a cell's parts list to recreate its metabolic activities in silico. Despite spectacular progress in genome sequencing, the parts lists for most organisms that we seek to manipulate remain highly incomplete, due to the dual problem of ‘unknown’ proteins and ‘orphan’ enzymes. The former are all the proteins deduced from genome sequence that have no known function, and the latter are all the enzymes described in the literature (and often catalogued in the EC database) for which no corresponding gene has been reported. Unknown proteins constitute up to about half of the proteins in prokaryotic genomes, and much more than this in higher plants and animals. Orphan enzymes make up more than a third of the EC database. Attacking the ‘missing parts list’ problem is accordingly one of the great challenges for post-genomic biology, and a tremendous opportunity to discover new facets of life's machinery. Success will require a co-ordinated community-wide attack, sustained over years. In this attack, comparative genomics is probably the single most effective strategy, for it can reliably predict functions for unknown proteins and genes for orphan enzymes. Furthermore, it is cost-efficient and increasingly straightforward to deploy owing to a proliferation of databases and associated tools.

Roughly half the carbon that crop plants fix by photosynthesis is subsequently lost by respiration. Nonessential respiratory activity leading to unnecessary CO₂ release is unlikely to have been minimized by natural selection or crop breeding, and cutting this large loss could complement and reinforce the currently dominant yield-enhancement strategy of increasing carbon fixation. Until now, however, respiratory carbon losses have generally been overlooked by metabolic engineers and synthetic biologists because specific target genes have been elusive. We argue that recent advances are at last pinpointing individual enzyme and transporter genes that can be engineered to (1) slow unnecessary protein turnover, (2) replace, relocate, or reschedule metabolic activities, (3) suppress futile cycles, and (4) make ion transport more efficient, all of which can reduce respiratory costs. We identify a set of engineering strategies to reduce respiratory carbon loss that are now feasible and model how implementing these strategies singly or in tandem could lead to substantial gains in crop productivity.

Phytol from chlorophyll degradation can be phosphorylated to phytyl-phosphate and phytyl-diphosphate, the substrate for tocopherol (vitamin E) synthesis. A candidate for the phytyl-phosphate kinase from Arabidopsis thaliana (At1g78620) was identified via a phylogeny-based approach. This gene was designated VITAMIN E DEFICIENT6 (VTE6) because the leaves of the Arabidopsis vte6 mutants are tocopherol deficient. The vte6 mutant plants are incapable of photoautotrophic growth. Phytol and phytyl-phosphate accumulate, and the phytyl-diphosphate content is strongly decreased in vte6 leaves. Phytol feeding and enzyme assays with Arabidopsis and recombinant Escherichia coli cells demonstrated that VTE6 has phytyl-P kinase activity. Overexpression of VTE6 resulted in increased phytyl-diphosphate and tocopherol contents in seeds, indicating that VTE6 encodes phytyl-phosphate kinase. The severe growth retardation of vte6 mutants was partially rescued by introducing the phytol kinase mutation vte5. Double mutant plants (vte5 vte6) are tocopherol deficient and contain more chlorophyll, but reduced amounts of phytol and phytyl-phosphate compared with vte6 mutants, suggesting that phytol or phytyl-phosphate are detrimental to plant growth. Therefore, VTE6 represents the missing phytyl-phosphate kinase, linking phytol release from chlorophyll with tocopherol synthesis. Moreover, tocopherol synthesis in leaves depends on phytol derived from chlorophyll, not on de novo synthesis of phytyl-diphosphate from geranylgeranyl-diphosphate.

Unlike animals, plants produce their own B vitamins but may nonetheless suffer vitamin deficiencies under stress conditions, with wide-ranging metabolic consequ