Functional responses of methanogenic archaea to syntrophic growth.

Methanococcus maripaludis grown syntrophically with Desulfovibrio vulgaris was compared with M. maripaludis monocultures grown under hydrogen limitation using transcriptional, proteomic and metabolite analyses. These measurements indicate a decrease in transcript abundance for energy-consuming biosynthetic functions in syntrophically grown M. maripaludis, with an increase in transcript abundance for genes involved in the energy-generating central pathway for methanogenesis. Compared with growth in monoculture under hydrogen limitation, the response of paralogous genes, such as those coding for hydrogenases, often diverged, with transcripts of one variant increasing in relative abundance, whereas the other was little changed or significantly decreased in abundance. A common theme was an apparent increase in transcripts for functions using H(2) directly as reductant, versus those using the reduced deazaflavin (coenzyme F(420)). The greater importance of direct reduction by H(2) was supported by improved syntrophic growth of a deletion mutant in an F(420)-dependent dehydrogenase of M. maripaludis. These data suggest that paralogous genes enable the methanogen to adapt to changing substrate availability, sustaining it under environmental conditions that are often near the thermodynamic threshold for growth. Additionally, the discovery of interspecies alanine transfer adds another metabolic dimension to this environmentally relevant mutualism

Analysis of the Arabidopsis cytosolic proteome highlights subcellular partitioning of central plant metabolism

International audienc

Modular Engineering of l-Tyrosine Production in Escherichia coli

Efficient biosynthesis of l-tyrosine from glucose is necessary to make biological production economically viable. To this end, we designed and constructed a modular biosynthetic pathway for l-tyrosine production in E. coli MG1655 by encoding the enzymes for converting erythrose-4-phosphate (E4P) and phosphoenolpyruvate (PEP) to l-tyrosine on two plasmids. Rational engineering to improve l-tyrosine production and to identify pathway bottlenecks was directed by targeted proteomics and metabolite profiling. The bottlenecks in the pathway were relieved by modifications in plasmid copy numbers, promoter strength, gene codon usage, and the placement of genes in operons. One major bottleneck was due to the bifunctional activities of quinate/shikimate dehydrogenase (YdiB), which caused accumulation of the intermediates dehydroquinate (DHQ) and dehydroshikimate (DHS) and the side product quinate; this bottleneck was relieved by replacing YdiB with its paralog AroE, resulting in the production of over 700 mg/liter of shikimate. Another bottleneck in shikimate production, due to low expression of the dehydroquinate synthase (AroB), was alleviated by optimizing the first 15 codons of the gene. Shikimate conversion to l-tyrosine was improved by replacing the shikimate kinase AroK with its isozyme, AroL, which effectively consumed all intermediates formed in the first half of the pathway. Guided by the protein and metabolite measurements, the best producer, consisting of two medium-copy-number, dual-operon plasmids, was optimized to produce >2 g/liter l-tyrosine at 80% of the theoretical yield. This work demonstrates the utility of targeted proteomics and metabolite profiling in pathway construction and optimization, which should be applicable to other metabolic pathways