One carbon metabolism in anaerobic bacteria: Regulation of carbon and electron flow during organic acid production

This reporting period, progress is reported on the following: metabolic pathway of solvent production in B. methylotrophicum; the biochemical mechanism for metabolic regulation of the succinate fermentation; models to understand the physiobiochemical function of formate metabolism in anaerobes and; models for understanding the influence of low pH on one carbon metabolism. (CBS

Laboratory evolution of Pyrococcus furiosus alcohol dehydrogenase to improve the production of (2S,5S)-hexanediol at moderate temperatures

There is considerable interest in the use of enantioselective alcohol dehydrogenases for the production of enantio- and diastereomerically pure diols, which are important building blocks for pharmaceuticals, agrochemicals and fine chemicals. Due to the need for a stable alcohol dehydrogenase with activity at low-temperature process conditions (30°C) for the production of (2S,5S)-hexanediol, we have improved an alcohol dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus (AdhA). A stable S-selective alcohol dehydrogenase with increased activity at 30°C on the substrate 2,5-hexanedione was generated by laboratory evolution on the thermostable alcohol dehydrogenase AdhA. One round of error-prone PCR and screening of ∼1,500 mutants was performed. The maximum specific activity of the best performing mutant with 2,5-hexanedione at 30°C was tenfold higher compared to the activity of the wild-type enzyme. A 3D-model of AdhA revealed that this mutant has one mutation in the well-conserved NADP(H)-binding site (R11L), and a second mutation (A180V) near the catalytic and highly conserved threonine at position 183

Industrial Systems Biology of Saccharomyces cerevisiae Enables Novel Succinic Acid Cell Factory.

Saccharomyces cerevisiae is the most well characterized eukaryote, the preferred microbial cell factory for the largest industrial biotechnology product (bioethanol), and a robust commerically compatible scaffold to be exploitted for diverse chemical production. Succinic acid is a highly sought after added-value chemical for which there is no native pre-disposition for production and accmulation in S. cerevisiae. The genome-scale metabolic network reconstruction of S. cerevisiae enabled in silico gene deletion predictions using an evolutionary programming method to couple biomass and succinate production. Glycine and serine, both essential amino acids required for biomass formation, are formed from both glycolytic and TCA cycle intermediates. Succinate formation results from the isocitrate lyase catalyzed conversion of isocitrate, and from the alpha-keto-glutarate dehydrogenase catalyzed conversion of alpha-keto-glutarate. Succinate is subsequently depleted by the succinate dehydrogenase complex. The metabolic engineering strategy identified included deletion of the primary succinate consuming reaction, Sdh3p, and interruption of glycolysis derived serine by deletion of 3-phosphoglycerate dehydrogenase, Ser3p/Ser33p. Pursuing these targets, a multi-gene deletion strain was constructed, and directed evolution with selection used to identify a succinate producing mutant. Physiological characterization coupled with integrated data analysis of transcriptome data in the metabolically engineered strain were used to identify 2nd-round metabolic engineering targets. The resulting strain represents a 30-fold improvement in succinate titer, and a 43-fold improvement in succinate yield on biomass, with only a 2.8-fold decrease in the specific growth rate compared to the reference strain. Intuitive genetic targets for either over-expression or interruption of succinate producing or consuming pathways, respectively, do not lead to increased succinate. Rather, we demonstrate how systems biology tools coupled with directed evolution and selection allows non-intuitive, rapid and substantial re-direction of carbon fluxes in S. cerevisiae, and hence show proof of concept that this is a potentially attractive cell factory for over-producing different platform chemicals

The Populus holobiont: dissecting the effects of plant niches and genotype on the microbiome

Background: Microorganisms serve important functions within numerous eukaryotic host organisms. An understanding of the variation in the plant niche-level microbiome, from rhizosphere soils to plant canopies, is imperative to gain a better understanding of how both the structural and functional processes of microbiomes impact the health of the overall plant holobiome. Using Populus trees as a model ecosystem, we characterized the archaeal/bacterial and fungal microbiome across 30 different tissue-level niches within replicated Populus deltoides and hybrid Populus trichocarpa × deltoides individuals using 16S and ITS2 rRNA gene analyses. Results: Our analyses indicate that archaeal/bacterial and fungal microbiomes varied primarily across broader plant habitat classes (leaves, stems, roots, soils) regardless of plant genotype, except for fungal communities within leaf niches, which were greatly impacted by the host genotype. Differences between tree genotypes are evident in the elevated presence of two potential fungal pathogens, Marssonina brunnea and Septoria sp., on hybrid P. trichocarpa × deltoides trees which may in turn be contributing to divergence in overall microbiome composition. Archaeal/bacterial diversity increased from leaves, to stem, to root, and to soil habitats, whereas fungal diversity was the greatest in stems and soils. Conclusions: This study provides a holistic understanding of microbiome structure within a bioenergy relevant plant host, one of the most complete niche-level analyses of any plant. As such, it constitutes a detailed atlas or map for further hypothesis testing on the significance of individual microbial taxa within specific niches and habitats of Populus and a baseline for comparisons to other plant species

Metabolic Engineering of Cofactor F420 Production in Mycobacterium smegmatis

Cofactor F420 is a unique electron carrier in a number of microorganisms including Archaea and Mycobacteria. It has been shown that F420 has a direct and important role in archaeal energy metabolism whereas the role of F420 in mycobacterial metabolism has only begun to be uncovered in the last few years. It has been suggested that cofactor F420 has a role in the pathogenesis of M. tuberculosis, the causative agent of tuberculosis. In the absence of a commercial source for F420, M. smegmatis has previously been used to provide this cofactor for studies of the F420-dependent proteins from mycobacterial species. Three proteins have been shown to be involved in the F420 biosynthesis in Mycobacteria and three other proteins have been demonstrated to be involved in F420 metabolism. Here we report the over-expression of all of these proteins in M. smegmatis and testing of their importance for F420 production. The results indicate that co–expression of the F420 biosynthetic proteins can give rise to a much higher F420 production level. This was achieved by designing and preparing a new T7 promoter–based co-expression shuttle vector. A combination of co–expression of the F420 biosynthetic proteins and fine-tuning of the culture media has enabled us to achieve F420 production levels of up to 10 times higher compared with the wild type M. smegmatis strain. The high levels of the F420 produced in this study provide a suitable source of this cofactor for studies of F420-dependent proteins from other microorganisms and for possible biotechnological applications

Archaic chaos: intrinsically disordered proteins in Archaea

Background: Many proteins or their regions known as intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) lack unique 3D structure in their native states under physiological conditions yet fulfill key biological functions. Earlier bioinformatics studies showed that IDPs and IDRs are highly abundant in different proteomes and carry out mostly regulatory functions related to molecular recognition and signal transduction. Archaea belong to an intriguing domain of life whose members, being microbes, are characterized by a unique mosaic-like combination of bacterial and eukaryotic properties and include inhabitants of some of the most extreme environments on the planet. With the expansion of the archaea genome data (more than fifty archaea species from five different phyla are known now), and with recent improvements in the accuracy of intrinsic disorder prediction, it is time to re-examine the abundance of IDPs and IDRs in the archaea domain.Results: The abundance of IDPs and IDRs in 53 archaea species is analyzed. The amino acid composition profiles of these species are generally quite different from each other. The disordered content is highly species-dependent. Thermoproteales proteomes have 14% of disordered residues, while in Halobacteria, this value increases to 34%. In proteomes of these two phyla, proteins containing long disordered regions account for 12% and 46%, whereas 4% and 26% their proteins are wholly disordered. These three measures of disorder content are linearly correlated with each other at the genome level. There is a weak correlation between the environmental factors (such as salinity, pH and temperature of the habitats) and the abundance of intrinsic disorder in Archaea, with various environmental factors possessing different disorder-promoting strengths. Harsh environmental conditions, especially those combining several hostile factors, clearly favor increased disorder content. Intrinsic disorder is highly abundant in functional Pfam domains of the archaea origin. The analysis based on the disordered content and phylogenetic tree indicated diverse evolution of intrinsic disorder among various classes and species of Archaea.Conclusions: Archaea proteins are rich in intrinsic disorder. Some of these IDPs and IDRs likely evolve to help archaea to accommodate to their hostile habitats. Other archaean IDPs and IDRs possess crucial biological functions similar to those of the bacterial and eukaryotic IDPs/IDRs