The global economy heads for a severe energy crisis: whereas the energy demand is going to rise, easily accessible sources of crude oil are expected to be depleted in only 10-20 years. Since a serious decline of oil supply and an associated collapse of the economy might be reality very soon, alternative energies and also biofuels that replace fossil fuels must be established. In addition, these alternatives should not further impair the environment and climate. About 90% of the biofuel market is currently captured by bioethanol and biodiesel. Biodiesel is composed of fatty acid alkyl esters (FAAE) and can be synthesized by chemical, enzymatic, or in vivo catalysis mainly from renewable resources. Biodiesel is already established as it is compatible with the existing fuel infrastructure, non-toxic, and has superior combustion characteristics than fossil diesel; and in 2008, the global production was 12.2 million tons. The biotechnological production of FAAE from low cost and abundant feedstocks like biomass will enable an appreciable substitution of petroleum diesel. To overcome high costs for immobilized enzymes, the in vivo synthesis of FAAE using bacteria represents a promising approach. This article points to the potential of different FAAE as alternative biofuels, e.g., by comparing their fuel properties. In addition to conventional production processes, this review presents natural and genetically engineered biological systems capable of in vivo FAAE synthesis.
The latex-clearing protein (Lcp K30 ) from the rubber-degrading bacterium Streptomyces sp. strain K30 is involved in the cleavage of poly(cis-1,4-isoprene), yielding isoprenoid aldehydes and ketones. Lcp homologues have so far been detected in all investigated clearing-zone-forming rubber-degrading bacteria. Internal degenerated oligonucleotides derived from lcp genes of Streptomyces sp. strain K30 (lcp K30 ), Streptomyces coelicolor strain A3(2), and Nocardia farcinica strains IFM10152 and E1 were applied in PCR to investigate whether lcp homologues occur also in the non-clearing-zone-forming rubber-utilizing bacteria Gordonia polyisoprenivorans strains VH2 and Y2K, Gordonia alkanivorans strain 44187, and Gordonia westfalica strain Kb1, which grow adhesively on rubber. The 1,230-and 1,224-bp lcp-homologous genes from G. polyisoprenivorans strain VH2 (lcp VH2 ) and G. westfalica strain Kb1 (lcp Kb1 ) were obtained after screening genomic libraries by degenerated PCR amplification, and their translational products exhibited 50 and 52% amino acid identity, respectively, to Lcp K30 . Recombinant lcp VH2 and lcp Kb1 harboring cells of the non-rubber-degrading Streptomyces lividans strain TK23 were able to form clearing zones and aldehydes on latex overlay-agar plates, thus indicating that lcp VH2 and lcp Kb1 encode functionally active proteins. Analysis by gel permeation chromatography demonstrated lower polymer concentrations and molecular weights of the remaining polyisoprenoid molecules after incubation with these recombinant S. lividans strains. Reverse transcription-PCR analysis demonstrated that lcp VH2 was transcribed in cells of G. polyisoprenivorans strain VH2 cultivated in the presence of poly(cis-1,4-isoprene) but not in the presence of sodium acetate. Anti-Lcp K30 immunoglobulin Gs, which were raised in this study, were rather specific for Lcp K30 and did not cross-react with Lcp VH2 and Lcp Kb1 . A lcp VH2 disruption mutant was still able to grow with poly(cis-1,4-isoprene) as sole carbon source; therefore, lcp VH2 seems not to be essential for rubber degradation in G. polyisoprenivorans.
The Gram-negative facultative chemolithoautotrophic bacterium Ralstonia eutropha strain H16 is known for its narrow carbohydrate utilization range, which limits its use for biotechnological production of polyhydroxyalkanoates and possibly other products from renewable resources. To broaden its substrate utilization range, which is for carbohydrates and related compounds limited to fructose, N-acetylglucosamine, and gluconate, strain H16 was engineered to use mannose and glucose as sole carbon sources for growth. The genes for a facilitated diffusion protein (glf) from Zymomonas mobilis and for a glucokinase (glk), mannofructokinase (mak), and phosphomannose isomerase (pmi) from Escherichia coli were alone or in combination constitutively expressed in R. eutropha strain H16 under the control of the neokanamycin or lac promoter, respectively, using an episomal broad-host-range vector. Recombinant strains harboring pBBR1MCS-3::glf::mak::pmi or pBBR1MCS-3::glf::pmi grew on mannose, whereas pBBR1MCS-3::glf::mak and pBBR1MCS-3::glf did not confer the ability to utilize mannose as a carbon source to R. eutropha. The recombinant strain harboring pBBR1MCS-3::glf::pmi exhibited slower growth on mannose than the recombinant strain harboring pBBR1MCS-3::glf::mak::pmi. These data indicated that phosphomannose isomerase is required to convert mannose-6-phosphate into fructose-6-phosphate for subsequent catabolism via the Entner-Doudoroff pathway. In addition, all plasmids also conferred to R. eutropha the ability to grow in the presence of glucose. The best growth was observed with a recombinant R. eutropha strain harboring plasmid pBBR1MCS-2::P nk ::glk::glf. In addition, expression of the respective enzymes was demonstrated at the transcriptional and protein levels and by measuring the activities of mannofructokinase (0.622 ؎ 0.063 U mg ؊1 ), phosphomannose isomerase (0.251 ؎ 0.017 U mg ؊1 ), and glucokinase (0.518 ؎ 0.040 U mg ؊1 ). Cells of recombinant strains of R. eutropha synthesized poly(3-hydroxybutyrate) to ca. 65 to 67% (wt/wt) of the cell dry mass in the presence of 1% (wt/vol) glucose or mannose as the sole carbon sources.The facultative chemolithoautotrophic Gram-negative Ralstonia eutropha strain H16, formerly known as Alcaligenes eutrophus, serves as model organism for studying the metabolism of poly(3-hydroxybuyrate) (PHB) and hydrogen-based chemolithoautotrophy. If grown autotrophically, strain H16 assimilates CO 2 via the Calvin-Benson-Bassham cycle. It uses also various organic compounds as a carbon and energy source if H 2 is absent. Under unbalanced growth conditions, PHB is accumulated in granules as a storage compound for carbon and energy (8). This is the case when a carbon source is abundant and if another macroelement such as nitrogen is lacking. The well-understood physiology of this "Knallgas" bacterium, its ability to be cultivated to high cell densities also at large scale, and the enormous polyhydroxyalkanoate (PHA) content of the cells led the industry consider to producing PHAs by fermentatio...
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