白色生物技术在可持续大规模化学生产中的应用

目前几乎所有有机化学品和塑料是从原油和天然气中生产的, 而生物技术的应用使得利用可再生资源进行大规模化工生产成为可能。以下主要综述了白色生物技术, 即利用细菌、酵母或酶将可发酵糖转化为特定的化学产品的技术。白色生物技术极大节省了不可再生能源的消耗, 减少了温室气体的排放。在有利条件下, 如果化工生产中相关技术有了发展并且可以成功以木质纤维素为原料, 那么到2050年不可再生能源的消耗将减少将近2/3 (67%)。欧洲（EU-25）地区的分析表明, 白色生物技术相关的用地在未来几年的欧洲不会受到制约, 尤其是有大量闲置资源的东欧。另外, 虽然原则上可以在白色生物技术中使用自然的细菌和酶, 但是很多专家认为, 利用经遗传改造生物体(GMO)可以达到高产量、高浓度、高效率, 这对实现经济活力是必要的。值得注意的是, 目前并不是所有的重组基因和其他物种间的相互作用所带来的后果都可预见, 因此化工生产释放的GMOs的安全失活和处理非常重要, 但是如果采取足够的预防措施, 在白色生物技术中应用GMOs的风险是可以控制的。我们认为, 生物生产过程的技术突破、下游生产过程的控制、化石燃料的高价格、可发酵糖的低价获得是生物质化学产业发展中的关键因素, 这4个因素及其他伴随策略是发展整体白色生物技术的要求。     

Invasion status of Florida bass Micropterus floridanus (Lesueur, 1822) in South Africa

Largemouth bass Micropterus salmoides are a popular North American angling species that was introduced into South Africa in 1928. To enhance the largemouth bass fisheries, Florida bass Micropterus floridanus were introduced into KwaZulu Natal, South Africa, in 1980. Knowledge on the status of M. floridanus in South Africa is required, because it lives longer and reaches larger sizes than M. salmoides, which may result in heightened impacts on native biota. Because M. floridanus are morphologically similar, but genetically distinct from M. salmoides, the distribution of this species was assessed by genetically screening 185 Micropterus sp. individuals sampled from 20 localities across South Africa using the mitochondrial ND2 gene. Individuals with mitochondrial DNA matching M. salmoides were recovered from 16 localities, whereas M. floridanus mitochondrial DNA was recovered from 13 localities. At nine localities (45%), the mitochondrial DNA of both species was detected. These results demonstrate M. floridanus dispersal to multiple sites across South Africa.     

Restriction barrier composed of an extracellular nuclease and restriction endonuclease in the unicellular cyanobacterium Microcystis sp.

Abstract The unicellular cyanobacterium Microcystis aeruginosa K-81 has two types of restriction barrier, an extracellular nuclease and sequence-specific endonuclease. The nuclease was detected in the culture supernatant and it was easily released from the cells by washing with water or buffer containing Triton X-100. This nuclease was identified as a polypeptide of about 28 kDa that digested covalently closed circular and linear double-stranded DNAs, including chromosomal DNA from M. aeruginosa K-81. Among another 13 Microcystis strains examined, 3 produced an extracellular nuclease. Furthermore, M. aeruginosa K-81 contained two sequence-specific endonucleases, Mae K81I and Mae K81II, which were isoschizomers of Sp /I and Sau 96I, respectively.     

Evolution of duplicate genes in a tetraploid animal, Xenopus laevis

To understand the evolution of duplicate genes, we compared rates of nucleotide substitution between 17 pairs of nonallelic duplicated genes in the tetraploid frog Xenopus laevis with rates between the orthologous loci of human and rodent. For all duplicated X. laevis genes, the number of synonymous substitutions per site (dS) was greater than the number of nonsynonymous substitutions per site (dN), indicating that these genes are subject to purifying selection. There was also a significant positive correlation (r = 0.915) between dN for the X. laevis genes and dN for the mammalian genes, suggesting that, at the amino acid level, the X. laevis genes and the mammalian genes are under similar constraints. Results of relative-rate tests showed nearly equal rates of nonsynonymous substitution in each copy of the X. laevis genes; apparently there are similar constraints on both copies. No correlation was found between dS for the X. laevis genes and dS for the mammalian genes. There was a significant positive correlation both between members of pairs of duplicated X. laevis genes (r = 0.951) and between human and rodent orthologues (r = 0.854) with respect to third- position G+C content but no such relationship between the X. laevis genes and either of their mammalian orthologues. The results indicate that both copies of a duplicate gene can be subject to purifying selection and thus support the hypothesis of selection against all genotypes containing a null allele at either of two duplicate loci.      

Inhibitory effects of auxins and related substances on the activity of an Arabidopsis glutathione S-transferase isozyme expressed in Escherichia coli

Glutathione S-transferases (GSTs; EC 2.5.1.18) are encoded by a gene family. Some GSTs have the capacity to bind to indole-3-acetic acid (IAA), whereas the gene expression of other GSTs is regulated by auxin. In order to assess a possible physiological significance of the auxin binding of GST, we investigated effects of auxins on the activity of GST expressed in Escherichia coli. cDNA cloning was carried out for the fifth gene ( GST5 ) of GST in Arabidopsis. Although the deduced amino acid sequence of GST5 was remotely related to that of the other Arabidopsis GSTs (less than 20% identical), the GST5 protein (GST5) expressed in E. coli showed GST activity. Apparent K_m values of GST5 are 0.86 and 1.29 m M for glutathione (GSH) and 1-chloro-2,4-dinitrobenzene, respectively. IAA, 2,4-dichlorophenoxyacetic acid (2,4-D), 1-naphthaleneacetic acid (1-NAA) and 2-NAA inhibited the enzyme activity competitively with respect to GSH. The apparent K_i of IAA is 1.56 m M . Salicylic acid inhibited GST activity in a noncompetitive manner. 2,4-D was the most inhibitory among the tested chemicals. GST5 bound to GSH-immobilized agarose gel was effectively eluted by IAA. These results indicate that IAA and the related substances bind to GST5 at the GSH-binding site, and exclude the possibility that the compounds could be substrates for GST5. Although the K_i value of IAA is too high for any physiological consequences, it might be assumed that GST activity is modulated in vivo by an auxin-related substance(s). The steady-state level of the GST5 mRNA was increased by wounding, heat shock, and spraying buffer on the plant, but was not influenced by auxin treatment.     

Construction of a novel suicide vector: selection for Escherichia coli HB101 recombinants carrying the DNA insert.

We constructed a new type of cloning vector, pERISH2, that transforms Escherichia coli HB101 only when a foreign DNA fragment is ligated into the cloning site of the plasmid vector. Plasmid pERISH2 carries the rcsB gene which is derived from the chromosome of E. coli HB101 and is involved in the regulation of colanic acid production. When E. coli HB101 is transformed by this vector carrying the intact rcsB gene, the gene product RcsB blocks bacterial growth. However, if the rcsB gene is inactivated by the insertion of a foreign DNA fragment, this recombinant plasmid no longer inhibits the growth of E. coli HB101. Although E. coli HB101 is not stably transformed by pERISH2, E. coli K-12 strains such as JM109 and C600 can harbor this vector. Therefore, pERISH2 can be amplified in JM109 and be prepared from this strain in a large quantity using conventional methods. A chromosomal gene library of Klebsiella pneumoniae is constructed easily and efficiently by the utilization of this new cloning vector.     

Immunochemical identity of peroxisomal enoyl-CoA hydratase with the peroxisome-proliferation -associated 80,000 mol wt polypeptide in rat liver

Peroxisome proliferators, which induce proliferation of hepatic peroxisomes, have been shown previously to cause a marked increase in an 80,000 mol wt polypeptide predominantly in the light mitochondrial and microsomal fractions of liver of rodents. We now present evidence to show that this hepatic peroxisome-proliferation-associated polypeptide, referred to as polypeptide PPA-80, is immunochemically identical with the multifunctional peroxisome protein displaying heat-labile enoyl-CoA hydratase activity. This conclusion is based on the following observations: (a) the purified polypeptide PPA-80 and the heat- labile enoyl-CoA hydratase from livers of rats treated with the peroxisome proliferators Wy-14,643 {[4-chloro-6(2,3-xylidino)-2-pyrimidinylthio]acetic acid} exhibit identical minimum molecular weights of approximately 80,000 on SDS polyacrylamide gel electrophoresis; (b) these two proteins are immunochemically identical on the basis of ouchterlony double diffusion, immunotitration, rocket immunoelectrophoresis, and crossed immunoelectrophoresis analysis; and (c) the immunoprecipitates formed by antibodies to polypeptide PPA-80 when dissociated on a sephadex G-200 column yield enoyl-CoA hydratase activity. Whether the polypeptide PPA-80 exhibits the activity of other enzyme(s) of the peroxisomal β-oxidation system such as fatty acyl-CoA oxidase activity or displays immunochemical identity with such enzymes remains to be determined. The availability of antibodies to polypeptide PPA-80 and enoyl-CoA hydratase facilitated immunofluorescent and immunocytochemical localization of the polypeptide PPA- 80 and enoyl-CoA hydratase in the rat liver. The indirect immunofluorescent studies with these antibodies provided direct visual evidence for the marked induction of polypeptide PPA-80 and enoyl-CoA hydratase in the livers of rats treated with Wy-14,643. The present studies also provide immunocytochemical evidence for the localization of polypeptide PPA- 80 and the heat-labile enoyl-CoA hydratase in the peroxisome, but not in the mitochondria, of hepatic parenchymal cells. These studies, therefore, provide morphological evidence for the existence of fatty acyl-CoA oxidizing system in peroxisomes. An increase of polypeptide PPA-80 on SDS polyacrylamide gel electrophoretic analysis of the subcellular fractions of liver of rodents treated with lipid-lowering drugs should serve as a reliable and sensitive indicator of enhanced peroxisomal β- oxidation system.     

A scaffoldless technique for self-generation of three-dimensional keratinospheroids on liquid crystal surfaces

We describe a new scaffold-free three-dimensional (3D) cell culture model using cholesteryl ester based lyotropic liquid crystal (LC) substrates. Keratinocytes were deposited randomly on the LC surface where they self-assembled into 3D microtissues or keratinospheroids. The cell density required to form spheroids was optimized. We investigated cell viability using dead/live cell assays. The adhesion characteristics of cells within the microtissues were determined using histological sectioning and immunofluorescence staining. Fourier transform infrared spectroscopy (FTIR) was used to characterize the biochemistry of the keratinospheroids. We found that both cells and microtissues could migrate on the LC surface. The viability study indicated approximately 80% viability of cells in the microtissues up to 20 days of culture. Strong intercellular adhesion was observed in the stratification of the multi-layered microspheroids using field emission-scanning electron microscopy (FE-SEM) and histochemical staining. The cytoskeleton and vinculins of the cells in the microtissues were expressed diffusely, but the microtissues were enriched with lipids and nucleic acids, which indicates close resemblance to the conditions in vivo. The basic 3D culture model based on LC may be used for cell and microtissue migration studies in response to cytochemical treatment.