Biotransformation of limonene by bacteria, fungi, yeasts, and plants

The past 5 years have seen significant progress in the field of limonene biotransformation, especially with regard to the regiospecificity of microbial biocatalysts. Whereas earlier only regiospecific biocatalysts for the 1,2 position (limonene-1,2-diol) and the 8-position (¿±-terpineol) were available, recent reports describe microbial biocatalysts specifically hydroxylating the 3-position (isopiperitenol), 6-position (carveol and carvone), and 7-position (perillyl alcohol, perillylaaldehyde, and perillic acid). The present review also includes the considerable progress made in the characterization of plant P-450 limonene hydroxylases and the cloning of the encoding genes

Prevalence of alkane monooxygenase genes in Arctic and Antarctic hydrocarbon-contaminated and pristine soils

The prevalence of four alkane monooxygenase genotypes (Pseudomonas putida GPo1, Pp alkB; Rhodococcus sp. strain Q15, Rh alkB1 and Rh alkB2; and Acinetobacter sp. strain ADP-1, Ac alkM) in hydrocarbon-contaminated and pristine soils from the Arctic and Antarctica were determined by both culture-independent (PCR hybridization analyses) and culture-dependent (colony hybridization analyses) molecular methods, using oligonucleotide primers and DNA probes specific for each of the alk genotypes. PCR hybridization of total soil community DNA detected the rhodococcal alkB genotypes in most of the contaminated (Rh alkB1, 18/20 soils; Rh alkB2, 13/20) and many pristine soils (Rh alkB1, 9/10 soils; Rh alkB2, 7/10), while Pp alkB was generally detected in the contaminated soils (15/20) but less often in pristine soils (5/10). Ac alkM was rarely detected in the soils (1/30). The colony hybridization technique was used to determine the prevalence of each of the alk genes and determine their relative abundance in culturable cold-adapted (5°C) and mesophilic populations (37°C) from eight of the polar soils. The cold-adapted populations, in general, possessed relatively higher percentages of the Rh alkB genotypes (Rh alkB1, 1.9% (0.55); Rh alkB2, 2.47% (0.89)), followed by the Pp alkB (1.13% (0.50)), and then the Ac alkM (0.53% (0.36)). The Rh alkB1 genotype was clearly more prevalent in culturable cold-adapted bacteria (1.9% (0.55)) than in culturable mesophiles (0.41 (0.55)), suggesting that cold-adapted bacteria are the predominant organisms possessing this genotype. Overall, these results indicated that (i) Acinetobacter spp. are not predominant members of polar alkane degradative microbial communities, (ii) Pseudomonas spp. may become enriched in polar soils following contamination events, and (iii) Rhodococcus spp. may be the predominant alkane-degradative bacteria in both pristine and contaminated polar soil

Differential protein expression during growth on linear versus branched alkanes in the obligate marine hydrocarbon‐degrading bacterium Alcanivorax borkumensis SK2T

Alcanivorax borkumensis SK2T is an important obligate hydrocarbonoclastic bacterium (OHCB) that can dominate microbial communities following marine oil spills. It possesses the ability to degrade branched alkanes which provides it a competitive advantage over many other marine alkane degraders that can only degrade linear alkanes. We used LC–MS/MS shotgun proteomics to identify proteins involved in aerobic alkane degradation during growth on linear (n‐C14) or branched (pristane) alkanes. During growth on n‐C14, A. borkumensis expressed a complete pathway for the terminal oxidation of n‐alkanes to their corresponding acyl‐CoA derivatives including AlkB and AlmA, two CYP153 cytochrome P450s, an alcohol dehydrogenase and an aldehyde dehydrogenase. In contrast, during growth on pristane, an alternative alkane degradation pathway was expressed including a different cytochrome P450, an alcohol oxidase and an alcohol dehydrogenase. A. borkumensis also expressed a different set of enzymes for β‐oxidation of the resultant fatty acids depending on the growth substrate utilized. This study significantly enhances our understanding of the fundamental physiology of A. borkumensis SK2T by identifying the key enzymes expressed and involved in terminal oxidation of both linear and branched alkanes. It has also highlights the differential expression of sets of β‐oxidation proteins to overcome steric hinderance from branched substrates

Harnessing plant biomass for biofuels and biomaterials

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72519/1/j.1365-313X.2008.03512.x.pd

Establishment of new crops for the production of natural rubber.

Natural rubber is a unique biopolymer of strategic importance that, in many of its most significant applications, cannot be replaced by synthetic alternatives. The rubber tree Hevea brasiliensis is the almost exclusive commercial source of natural rubber currently and alternative crops should be developed for several reasons, including: a disease risk to the rubber tree that could potentially decimate current production, a predicted shortage of natural rubber supply, increasing allergic reactions to rubber obtained from the Brazilian rubber tree and a general shift towards renewables. This review summarizes our knowledge of plants that can serve as alternative sources of natural rubber, of rubber biosynthesis and the scientific gaps that must be filled to bring the alternative crops into production

TOPOLOGY OF THE MEMBRANE-BOUND ALKANE HYDROXYLASE OF PSEUDOMONAS-OLEOVORANS

The Pseudomonas oleovorans alkane hydroxylase is an integral cytoplasmic membrane protein that is expressed and active in both Escherichia coli and P. oleovorans. Its primary sequence contains eight hydrophobic stretches that could span the membrane as alpha-helices. The topology of alkane hydroxylase was studied in E. coli using protein fusions linking different amino-terminal fragments of the alkane hydroxylase (AlkB) to alkaline phosphatase (PhoA) and to beta-galactosidase (LacZ). Four AlkB-PhoA fusions were constructed using transposon TnphoA. Site-directed mutagenesis was used to create PstI sites at 12 positions in AlkB. These sites were used to create AlkB-PhoA and AlkB-LacZ fusions. With respect to alkaline phosphatase and beta-galactosidase activity each set of AlkB-PhoA and AlkB-LacZ fusions revealed the expected complementary activities. At three positions, PhoA fusions were highly active, whereas the corresponding LacZ fusions were the least active. At all other positions the PhoA fusions were almost completely inactive, but the corresponding LacZ fusions were highly active. These data predict a model for alkane hydroxylase containing six transmembrane segments. In this model the amino terminus, two hydrophilic loops, and a large carboxyl-terminal domain are located in the cytoplasm. Only three very short loops near amino acid positions 52, 112, and 251 are exposed to the periplasm