Screening And Application Of Gradient Constitutive Promoters In Gluconobacter Oxydans

Gluconobacter oxydans is widely used in the production of value added product, such as 5-keto-D-gluconic acid, dihydroxyacetone and 2-keto-L-gulonic acid(precursor of vitamin C), due to its ability of rapid incomplete oxidation of many sugars and alcohols. This makes this strain have great potential for industrial production. A promoter, an important element for the regulation of gene transcription, is often used as an effective genetic tool in metabolic engineering of many strains. However, the study on the screening of pomoters in G. oxydans was insufficient, which limits the available promoters used in metabolic engineering of this industrial strain. In this study, based on the next generation high throughput RNA-Seq technology, a gradient of constitutive promoters with different strength were screened at a genome scale. The strongest Pdank promoter obtained here was further used to overexpress the key D-sorbitol dehydrogenase gene sld AB1 determined by RNA-Seq. The results demonstrated the high ability of this target promoter for gene transcription; Furthermore, the yield of L-sorbose synthesis was increased and the fermentation time was shortened dramatically due to this promoter. Main results were described as follows:(1) The gene expression levels of G. oxydans in 5 different growth phases were obtained by the RNA-Seq technology. The strain cells of 4 h, 8 h, 12 h, 20 h and 40 h in a 1-L fermenter were collected to construct the RNA-Seq libraries, respectively. High quality sequencing data were obtained and the gene transcription levels of whole genome were determined by FPKM(Fragments Per Kilobase of exon model per Million mapped reads) value after a series of bioinformatics analysis. The statistics results showed that the FPKM value of about 50% of the whole gene was between 32 and 320, and about 25% of the whole gene was in 320-10,000 and about 25% of the whole gene was in 1-32, with a few extremely high and low ones. Combined with genome sequencing information, RNA-Seq results also illustrated that the sld AB1 and sld SLC gene played a key role in the oxidization of D-sorbitol to L-sorbose.(2) Based on the RNA-Seq results, a gradient of constitutive promoters with different strength were screened including the strongest promoter Pdnak, demonstrated by the enhanced green fluorescent protein(egfp) report gene. Refer to the FPKM value of the whole transcript, a gradient of constitutive genes including dank, tuf and mdh in high, immediate and low transcription level were selected. The recombinant strains for the expression of egfp report genes under selected promoters were constructed. The transcription and expression levels of egfp in different recombinant strains were measured by quantitative real-time PCR(q RT-PCR) and fluorescence micro-plate reader, respectively. A gradient of constitutive promoters with high, immediate and low strength were screened, including the strongest promoter Pdnak. Take the Ptuf for reference, Pdnak and Phistone were promoters with high strength, Ptuf, Phistone and Poutmem were promoters with immediate strength, Psod and Phsp90 were promoters with low strength, the promoters’ strength can be described as an equation, Pdnak : Phistone : Ptuf : Poutmem : Psod : Pmdh : Phsp90 =10.0 : 2.5 : 1.0 : 0.9 : 0.8 : 0.2 : 0.1.(3) The ability of the strongest promoter Pdnak for gene transcription was demonstrated and the sld AB1 was overexpressed under the Pdnak promoter in G. oxydans. The strongest Pdnak promoter and control promoter Ptuf were selected to construct the recombinant strain G. oxydans p BBR-Pdnak-sld AB1 and G. oxydans p BBR-Ptuf-sld AB1, respectively. The transcription level of sld AB1 in G. oxydans p BBR-Pdnak-sld AB1 exhibited a 2.2-fold and 5.1-fold increase compared to G. oxydans p BBR-Ptuf-sld AB1 and the wild type strain G. oxydans WSH-003, respectively. Furthermore, the flask fermentation experiments showed that overexpression of sld AB1 by Pdank in G. oxydans p BBR-Pdnak-sld AB1 shortened the fermentation time of L-sorbose synthesis from D-sorbitol by 33.3% and enhanced the yield of L-sorbose by 11.2%, respectively, compared with the wild type strain.