| Sophorolipids is a biosurfactant produced by yeast.Compared with chemical surfactants,sophorolipids are low-toxic or non-toxic,have good environmental compatibility,and have good dispersion,emulsification and solubilization properties.In addition,it has the characteristics of anti-tumor,anti-virus and inhibition of leukocyte growth.Good characteristics make the sophorolipids widely used in daily chemical,petroleum,environmental protection,medicine and other fields.At present,the main factor limiting the large-scale fermentation of sophorolipids is that the fermentation cost is too high.The key factor limiting its cost reduction is that the low supply and utilization level of oxygen in the fermentation process leads to low utilization of oil-soluble substrate and long fermentation cycle.In the process of synthesizing sophorolipids,the hydroxylation of fatty acids is a key step in the synthesis of sophorolipids,which requires the consumption of large amounts of oxygen.During high-density fermentation,the level of dissolved oxygenis often a limiting factor.The level of dissolved oxygen outside the cells can be effectively increased by adjusting the fermentation conditions.It is feasible to improve the dissolved oxygen in the fermentation process by increasing the aeration rate,accelerating the stirring speed,and increasing the tank pressure.But these measures have high requirements on the strength of the equipment,and the excessive partial pressure of carbon dioxide also limits the growth of the cells.The literature show that vitreoscilla hemoglobin(VHb)can increase the utilization of oxygen by gene cloning bacteria at the molecular level,and its application can not only reduce oxygen and energy consumption,but also does not require additional equipment investment.In this study,the recombination technology in modern molecular biology was used to optimize the coding sequence of the vitreoscilla hemoglobin gene vgb according to the codon preference of Starmerella bombicola,making it more suitable for heterologous expression in S.bombicola.The expression vector pSBEX-htp-vgb was linearized by double-digested,and electroporated into S.bombicola O-13-1 strain.The positive recombinant-VHb~+strain was screened.The test of CO difference spectra indicate that the VHb gene is successfully expressed in the recombinant strain VHb~+and its product has biologically activity.The successful construction of recombinant strain containing VHb gene provides a good experimental materials for the reaserch of the effect of VHb on sophorolipid fermentation.The effect of VHb gene on the production of sophorolipids was studied.The results showed that the sophorolipid yield of the recombinant strain VHb~+was increased by 16.23%compared with the original strain VHb~-in the shaking bottle experiment.In 5L fermenter fermentation,under the oxygen-rich condition(rotation speed 400 rpm,ventilation 1.0 vvm),the biomass and sophorolipid production of the two strains were not significantly different,but in the synthesis stage of sophorolipids,the oxygen consumption of VHb~+strain was 21.8%lower than that of VHb~-strain;Under the oxygen-limited condition(rotation speed 350 rpm,aeration rate 1.0 vvm),the utilization of dissolved oxygen in the VHb~+strain was significantly improved during the fermentation,and the sophorolipid production was 25.1%higher than that of the VHb~-strain.The mechanism of the effect of VHb on the sophorolipid production and dissolved oxygen was studied by qPCR.The relative expression levels of key enzyme genes of the TCA cycle,respiratory chain and sophorolipid synthesis of the recombinant strain VHb~+were similar compared with the wide-type strain VHb~-at 24h.while at 72h(exponential growth period of biosynthesis of sophorolipid),the expression level of key enzyme genes of strain VHb~+in these three metabolisms was significantly increased,which indicated that the expression of VHb gene could increase the expression level of TCA cycle and electron transport chain,improve the utilization efficiency of oxygen,and promote the synthesis of sophorolipid.In summary,the heterologous expression of vgb enhances the utilization efficiency of oxygen in the strain O-13-1,increases the sophorolipids production under oxygen-poor conditions,and which is an effective method to alleviate the restriction of low oxygen supply on the fermentation of sophorolipids.The fermentation conditions of the recombinant strain VHb~+were optimized by the one-time-one-factor experiment,and the results show that these conditions favourable for the fermentation production of sophorolipid including rotational speed,aeration and tank pressure were 350 rpm,0.6 vvm and 0.04 Mpa,respectively.Under this condition,the sophorolipid production,biomass and dissolved oxygen in the fermentation broth are relatively high,and the final sophorolipid production is a transparent viscous liquid,which is easily separated by natural sedimentation.Therefore,it is of great significance to ensure appropriate dissolved oxygen in the fermentation liquid for increasing the yield of sophorolipids in industrial fermentation.The expression of the VHb gene vgb in strain O-13-1 increases the intracellular oxygen transfer rate and improves the oxygen utilization efficiency.This strategy provides a new way to solve the problem of insufficient oxygen supply during the fermentation process of sophorolipids,and supplies a basis for energy saving and consumption,and has a good application prospect. |
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