RecET recombination system driving chromosomal target gene replacement in Zymomonas mobilis

Background: Zymomonas mobilis is a Gram-negative microaerophilic bacterium with excellent ethanol-producing capabilities. The RecET recombination system provides an efficient tool for direct targeting of genes in the bacterial chromosome by PCR fragments. Results: The plasmids pSUZM2a-RecET and pSUZM2a-RecE588T were first developed to co-express RecE or RecE588 and RecT for homologous recombination. Thereafter, the PCR fragments of the tetracycline resistance marker gene flanked by 60 bp of adhA (alcohol dehydrogenase I) or adhB (alcohol dehydrogenase II) homologous sequences were electroporated directly into ZM4 cells harboring pSUZM2a-RecET or pSUZM2a-RecE588T. Both adhA and adhB were replaced by the tetracycline resistance gene in ZM4, yielding two mutant strains, Z. mobilis ZM4 ΔadhA and Z. mobilis ZM4 ΔadhB. These two mutants showed varying extent of reduction in ethanol production, biomass generation, and glucose metabolism. Furthermore, enzyme activity of alcohol dehydrogenase II in Z. mobilis ZM4 ΔadhB exhibited a significant reduction compared to that of wild-type ZM4. Conclusion: This approach provided a simple and useful method for introducing mutations and heterologous genes in the Z. mobilis genome.

Three new shuttle vectors for heterologous expression in Zymomonas mobilis

Background: Zymomonas mobilis, as a novel platform for bio-ethanol production, has been attracted more attention and it is very important to construct vectors for the efficient expression of foreign genes in this bacterium. Results: Three shuttle vectors ( pSUZM 1, pSUZM2 and pSUZM3 ) were first constructed with the origins of replication from the chromosome and two native plasmids (pZZM401 and pZZM402) of Z. mobilis ZM4, respectively. The three shuttle vectors were stable in Z. mobilis ZM4 and have 3,32 and 27 copies, respectively. The promoter Ppdc (a), from the pyruvate decarboxylase gene, was clonedinto the shuttle vectors, generatingthe expressionvectors pSUZM1(2, 3)a. The codon-optimized glucoamylase gene from Aspergillus awamori combined with the signal peptide sequence from the alkaline phosphatase gene of Z. mobilis was cloned into pSUZM1(2, 3)a, resulting in the plasmids pSUZM1a-GA, pSUZM2a-GA and pSUZM3a-GA, respectively. After transforming these plasmids into Z. mobilis ZM4, the host was endowed with glucoamylase activity for starch hydrolysis. Both pSUZM2a-GA and pSUZM3a-GA were more efficientatproducingglucoamylase thanpSUZM1a-GA. Conclusions: These results indicated that these expression vectors are useful tools for gene expression in Z. mobilis and this could provide a solid foundation for further studies of heterologous gene expression in Z. mobilis.

Optimizing expression and purification of an ATP-binding gene gsiA from Escherichia coli k-12 by using GFP fusion

The cloning, expression and purification of the glutathione (sulfur) import system ATP-binding protein (gsiA) was carried out. The coding sequence of Escherichia coli gsiA, which encodes the ATP-binding protein of a glutathione importer, was amplified by PCR, and then inserted into a prokaryotic expression vector pWaldo-GFPe harboring green fluorescent protein (GFP) reporter gene. The resulting recombinant plasmid pWaldo-GFP-GsiA was transformed into various E. coli strains, and expression conditions were optimized. The effect of five E. coli expression strains on the production of the recombinant gsiA protein was evaluated. E. coli BL21 (DE3) was found to be the most productive strain for GsiA-GFP fusion-protein expression, most of which was insoluble fraction. However, results from in-gel and Western blot analysis suggested that expression of recombinant GsiA in Rosetta (DE3) provides an efficient source in soluble form. By using GFP as reporter, the most suitable host strain was conveniently obtained, whereby optimizing conditions for overexpression and purification of the proteins for further functional and structural studies, became, not only less laborious, but also time-saving.

Genetic diversity and geographical differentiation of cultivated six-rowed naked barley landraces from the Qinghai-Tibet plateau of China detected by SSR analysis

Cultivated six-rowed naked barley (Hordeum vulgare ssp. hexastichon var. nudum Hsü) is the oldest cultivated barley in China. We used 35 simple sequence repeat (SSR) markers selected from seven barley linkage groups to study the genetic diversity, geographical differentiation and evolutionary relationships among 65 H. vulgare ssp. hexastichon landrace accessions collected from the Qinghai-Tibet plateau of China, 25 accessions from Tibet (TB), 20 from Qinghai (QH) and 20 from Ganzi (GZ) prefecture in Sichuan province. At the 35 SSR loci we identified 248 alleles among the 65 accessions, 119 (47.98 percent) of the alleles being common alleles. We also found that the TB accessions possessed 47 private alleles, about 1.5 times more than the 31 private alleles found in the QH accessions and about 5 times more than 9 private alleles found in the GZ accessions. Generally, the TB accessions showed significantly higher genetic diversity than either the QH or GZ accessions whereas no significant difference in genetic diversity was found between the QH and GZ accessions. Partitioning analysis of genetic diversity showed that about 81 percent of the total variation was due to within-subgroup diversity and about 19 percent was clearly accounted for by geographical differentiation among the three subgroups. The distributions of alleles for most loci (71.4 percent) were significantly different among the three subgroups and geographical differentiation could be found according to the distribution of SSR alleles. Cluster analysis indicated that most of the accessions could be clustered into groups which basically coincided with their geographical distribution. These results suggest that Tibet might be a center of genetic diversity for cultivated barley, the cultivated six-rowed naked barley on the Qinghai-Tibet plateau of China may have evolved in Tibet and spread to Qinghai and then to Ganzi prefecture of Sichuan province.