Lactic acid production by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing a <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> lactate dehydrogenase gene

This work demonstrates the first example of a fungal lactate dehydrogenase (LDH) expressed in yeast. A L(+)-LDH gene, ldhA, from the filamentous fungus Rhizopus oryzae was modified to be expressed under control of the Saccharomyces cerevisiae adh1 promoter and terminator and then placed in a 2μ-containing yeast-replicating plasmid. The resulting construct, pLdhA68X, was transformed and tested by fermentation analyses in haploid and diploid yeast containing similar genetic backgrounds. Both recombinant strains utilized 92 g glucose/l in approximately 30 h. The diploid isolate accumulated approximately 40% more lactic acid with a final concentration of 38 g lactic acid/l and a yield of 0.44 g lactic acid/g glucose. The optimal pH for lactic acid production by the diploid strain was pH 5. LDH activity in this strain remained relatively constant at 1.5 units/mg protein throughout the fermentation. The majority of carbon was still diverted to the ethanol fermentation pathway, as indicated by ethanol yields between 0.25–0.33 g/g glucose. S. cerevisiae mutants impaired in ethanol production were transformed with pLdhA68X in an attempt to increase the lactic acid yield by minimizing the conversion of pyruvate to ethanol. Mutants with diminished pyruvate decarboxylase activity and mutants with disrupted alcohol dehydrogenase activity did result in transformants with diminished ethanol production. However, the efficiency of lactic acid production also decreased. Electronic Publication     

酵母耐盐机制的研究进展

酵母是一种真核模式生物同时也是一种耐盐微生物,其基因表达和信号传导系统的调节机制及离子运输机制与高等真核生物类似。酵母耐盐机制的研究有助于阐明真核生物的耐盐机制。本文综述了酵母在盐胁迫下的信号传导途径和分子应答机制,以及在酵母耐盐机制研究中主要的研究方法。 Abstract:Yeast is a model eukoryotic organism and salt-tolerant microorganism.The regulative mechanism of gene expression and signal transduction and ion transport of yeast is similar to that of higher eukoryotic organism.The research on salt-tolerant mechanism of yeast will be helpful to the illustrate the salt-tolerant mechanism of higher eukoryotic organism.This review summarized the signal transduction pathway and molercular responses of yeast under salt stress and the major research methods in the research on the salt-tolerant mechenism in yeast.     

Discovering genes responsible for silk synthesis in Bombyx mori by piggyBac‐based random insertional mutagenesis

Silkworm mutants are valuable resources for both transgenic breeding and gene discovery. PiggyBac-based random insertional mutagenesis has been widely used in gene functional studies. In order to discover genes involved in silk synthesis, a piggyBac-based random insertional library was constructed using Bombyx mori, and the mutants with abnormal cocoon were particularly screened. By this means, a “thin cocoon” mutant was identified. This mutant revealed thinner cocoon shell and shorter posterior silk gland (PSG) compared with the wild type. The messenger RNA (mRNA) levels of all the three fibroin genes, including Fib-H, Fib-L and P25, were significantly down-regulated in the PSG of mutants. Four piggyBac insertion sites were identified in Aquaporin (AQP), Longitudinals lacking protein-like {Lola), Glutamyl aminopeptidase-like (GluAP) and Loc101744460. The mRNA levels of all the four genes were significantly altered in the silk gland of mutants. In particular, the mRNA amount of AQP, a gene responsible for the regulation of osmotic pressure, decreased dramatically immediately prior to the spinning stage in the anterior silk gland of mutants. The identification of the genes disrupted in the “thin cocoon” mutant in this study provided useful information for understanding silk production and transgenic breeding of silkworms in the future.     

Arabidopsis AtVPS15 plays essential roles in pollen germination possibly by interacting with AtVPS34

VPS 15 protein is a component of the phosphatidylinositol 3-kinase complex which plays a pivotal role in the development of yeast and mammalian cells.The knowledge about the function of its homologue in plants remains limited.Here we report that AtVPS15, a homologue of yeast VPS15p in Arabidopsis,plays an essential role in pollen germination.Homozygous T-DNA insertion mutants of AtVPS15 could not be obtained from the progenies of self-pollinated heterozygous mutants.Reciprocal crosses between atvpslS mutants and wild-type Arabidopsis revealed that the T-DNA insertion was not able to be transmitted by male gametophytes.DAPI staining, Alexander’s stain and scanning electron microscopic analysis showed that atvpsl5 heterozygous plants produced pollen grains that were morphologically indistinguishable from wild-type pollen,whereas in vitro germination experiments revealed that germination of the pollen grains was defective.GUS staining analysis of transgenic plants expressing the GUS reporter gene driven by the AtVPS15 promoter showed that AtVPSI5 was mainly expressed in pollen grains.Finally,DUALmembrane yeast two-hybrid analysis demonstrated that AtVPS15 might interact directly with AtVPS34.These results suggest that AtVPS15 is very important for pollen germination,possibly through modulation of the activity of PI3-kinase.     

Construction of brewing-wine Aspergillus oryzaepyrG- mutant bypyrG gene deletion and its application in homology transformation

pyrG- host cells are indispensable for pyrG- based transformation system. Isolations ofpyrG- host cells by random mutations are limited by time-consuming, unclear genetic background and potential interferences of homogenous recombination. The purpose of this study was to construct brewing-wine Aspergillus oryzae pyrG- mutant by sitedirected mutation of pyrG gene deletion which would be used as a host for further transformation, pMD-pyrGAB, a vector carrying pyrG deletion cassette, was used to con- struct pyrG- mutant of A. oryzae. Three stable pyrG deletion mutants of A. oryzae were isolated by resistant to 5-fluoroorotic acid and confirmed by polymerase chain reaction analysis, indicating thatpyrG was completely excised. The ApyrG mutants were applied as pyrG- host cells to disrupt xdh gene encoding xylitol dehydrogenase, which involves in xylitol production ofA. Oryzae. The xdh disrup- tion mutants were efficiently constructed by transforming a pMD-pyrC-xdh disruption plasmid carrying pyrG, and the produced xylitol concentration of the Axdh mutant was three times as much as that of the ApyrG recipient. Site-directed pyrG gene deletion is thus an effective way for the isolation of pyrG- host cells, and the established host-vector system could be applied in further functional genomics analysis and molecular breeding ofA. Oryzae.