Construction And Application Of Inducible Expression System For Streptococcus Zooepidemicus ATCC39920

Hyaluronic acid (HA) is a linear, high molecular weight polysaccharide and has been widely applied in medicine, cosmetics and other industries. Streptococcus zooepidemicus is used for microbial production of hyaluronan on an industrial scale. Intensive research on S. zooepidemicus biology and metabolism mechanism is of great significance. Ideal inducible promoter system is an effective way to study gene function. However, the lack of an effective inducible promoter system for S.zooepidemicus has notably prevented the functional genomics analysis of this gram-positive bacterium. In this study, Streptococcus equi subsp. zooepidemicus ATCC39920 was used as the object of study. Here, we report the development of four plasmid-based inducible promoter systems, that is, sucrose, lactose, xylose and nisin controlled gene expression system. hasA was used as report gene, which encoded hyaluronan synthase. Deletion of has A resulted in non-mucoid morphology, so AhasA was used as host strain. The feasibility of these systems was verified by detecting the capsule of AhasA. Then, we projected a controllable two stage culture strategy basing on the characteristic of AhasA/pLH201 to enhance HA production. The main results were as follows:1. Construction of stable plasmid for Streptococcus zooepidemicus ATCC39920plasmid pSET4s was used as based vector for creating E.coli-Streptococcus shuttle plasmid pLH200, which harbored rep A form pWV01 plasmid and spectinomycin resistant gene. S. zooepidemicus/pLH200 mutants were cultured for 24 h without antibiotic, which lost pLH200 at a frequency of 0%. This plasmid can be used for intensive research and resistance variation caused by the use of antibiotics may be avoided at the same time.2. Construction and analysis of four inducible promoter systemsplasmid pLH200 was used as based vector for creating inducible plasmids. scrR-PscrA cassette was amplified and ligated into pLH200 to generate sucrose inducible plasmid. has A, as reporter gene, was placed at the downstream of the PscrA promoter to make plasmid pLH200::scrR-PscrA::hasA(pLH201).scrR-PscrA cassette was replaced by lacR-PlacA, xylR-PxylA and nisk-nisR-PnisA to construct lactose, xylose and nisin controlled gene expression system, respectively. Feasibility analysis showed that:Lactose controlled inducible promoter system is an inefficient system for expressing targeted gene. Xylose and nisin controlled gene expression system are not working. Sucrose controlled gene expression system is efficient and rigorous, which will greatly promote the research of functional gene.3. Establish a controllable two-step fermentation mode to produce HATransformation of pLH201 plasmid into AhasA generated AhasA/pLH201 mutants. When cultured these mutants in sucrose-containing media, high levels of capsule were detected. While there did not detected capsule in the presence of the glucose, which relieved the limitation on the transmission of dissolved oxygen and substrate. A controllable two-step fermentation mode was enhanced basing on the feature of △hasA/pLH201. The strategy is, △hasA/pLH201 mutants are cultured with an initial glucose concentration of 30 g/L during 0-12 h to obtain high density cells and then further cultures are performed during 12-24 h with an initial sucrose concentration of 20 g/L to harvest HA. With the proposed two-stage culture strategy, HA production of AhasA/pLH201 reached at 3.4 g/L, while there did not detected HA of AhasA. Following further study, once high density of AhasA mutants were obtained, the production of HA will undoubted increase by this controllable two-step fermentation mode.