Characterization and assessment of two biocontrol bacteria against Pseudomonas syringae wilt in Solanum lycopersicum and its genetic responses.

Pseudomonas and Bacillus species are attractive due to their potential bio-control application against plant bacterial pathogens. Pseudomonas aeruginosa strain D4 and Bacillus stratosphericus strain FW3 were isolated from mine tailings in South Korea. In these potent bacterial strains, we observed improved antagonistic activity against Pseudomonas syringae DC3000. These strains produced biocatalysts for plant growth promotion, and in vivo examination of Solanum lycopersicum included analysis of disease severity, ion leakage, chlorophyll content, and H2O2 detection. In addition, regulation of the defense genes pathogen-related protein 1a (PR1a) and phenylalanine ammonia lyase (PAL) was compared with treated plants and untreated control plants. The results suggest that these two bacterial strains provide protection against plant pathogens via direct and indirect modes of action and could be used as a bio-control agent.

Genetically engineered probiotic for the treatment of phenylketonuria (PKU); assessment of a novel treatment in vitro and in the PAHenu2 mouse model of PKU.

Phenylketonuria (PKU) is a genetic disease characterized by the inability to convert dietary phenylalanine to tyrosine by phenylalanine hydroxylase. Given the importance of gut microbes in digestion, a genetically engineered microbe could potentially degrade some ingested phenylalanine from the diet prior to absorption. To test this, a phenylalanine lyase gene from Anabaena variabilis (AvPAL) was codon-optimized and cloned into a shuttle vector for expression in Lactobacillus reuteri 100-23C (pHENOMMenal). Functional expression of AvPAL was determined in vitro, and subsequently tested in vivo in homozygous PAHenu2 (PKU model) mice. Initial trials of two PAHenu2 homozygous (PKU) mice defined conditions for freeze-drying and delivery of bacteria. Animals showed reduced blood phe within three to four days of treatment with pHENOMMenal probiotic, and blood phe concentrations remained significantly reduced (P < 0.0005) compared to untreated controls during the course of experiments. Although pHENOMMenal probiotic could be cultured from fecal samples at four months post treatment, it could no longer be cultivated from feces at eight months post treatment, indicating eventual loss of the microbe from the gut. Preliminary screens during experimentation found no immune response to AvPAL. Collectively these studies provide data for the use of a genetically engineered probiotic as a potential treatment for PKU.

Development of a combined biological and chemical process for production of industrial aromatics from renewable resources.

Production of industrial aromatic chemicals from renewable resources could provide a competitive alternative to traditional chemical synthesis routes. This review describes the engineering of microorganisms for the production of p-hydroxycinnamic acid (pHCA) and p-hydroxystyrene (pHS) from glucose. The initial process concept was demonstrated using a tyrosine-producing Escherichia coli strain that overexpressed both fungal phenylalanine/tyrosine ammonia lyase (PAL) and bacterial pHCA decarboxylase (pdc) genes. Further development of this bioprocess resulted in uncoupling the pHCA and pHS production steps to mitigate their toxicity to the production host. The final process consists of a fermentation step to convert glucose to tyrosine using a tyrosine-overproducing E. coli strain. This step is followed by a single biotransformation reaction to deaminate tyrosine to pHCA through immobilized E. coli cells that overexpress the Rhodotorula glutinis PAL gene. Finally, chemical decarboxylation of pHCA produces pHS. This multifaceted approach, which integrates biology, chemistry, and engineering, has allowed development of an economical process at scales suitable for industrial applications.

An in silico assessment of gene function and organization of the phenylpropanoid pathway metabolic networks in Arabidopsis thaliana and limitations thereof.

The Arabidopsis genome sequencing in 2000 gave to science the first blueprint of a vascular plant. Its successful completion also prompted the US National Science Foundation to launch the Arabidopsis 2010 initiative, the goal of which is to identify the function of each gene by 2010. In this study, an exhaustive analysis of The Institute for Genomic Research (TIGR) and The Arabidopsis Information Resource (TAIR) databases, together with all currently compiled EST sequence data, was carried out in order to determine to what extent the various metabolic networks from phenylalanine ammonia lyase (PAL) to the monolignols were organized and/or could be predicted. In these databases, there are some 65 genes which have been annotated as encoding putative enzymatic steps in monolignol biosynthesis, although many of them have only very low homology to monolignol pathway genes of known function in other plant systems. Our detailed analysis revealed that presently only 13 genes (two PALs, a cinnamate-4-hydroxylase, a p-coumarate-3-hydroxylase, a ferulate-5-hydroxylase, three 4-coumarate-CoA ligases, a cinnamic acid O-methyl transferase, two cinnamoyl-CoA reductases) and two cinnamyl alcohol dehydrogenases can be classified as having a bona fide (definitive) function; the remaining 52 genes currently have undetermined physiological roles. The EST database entries for this particular set of genes also provided little new insight into how the monolignol pathway was organized in the different tissues and organs, this being perhaps a consequence of both limitations in how tissue samples were collected and in the incomplete nature of the EST collections. This analysis thus underscores the fact that even with genomic sequencing, presumed to provide the entire suite of putative genes in the monolignol-forming pathway, a very large effort needs to be conducted to establish actual catalytic roles (including enzyme versatility), as well as the physiological function(s) for each member of the (multi)gene families present and the metabolic networks that are operative. Additionally, one key to identifying physiological functions for many of these (and other) unknown genes, and their corresponding metabolic networks, awaits the development of technologies to comprehensively study molecular processes at the single cell level in particular tissues and organs, in order to establish the actual metabolic context.