| Farnesene is a sesquiterpene compound composed of three isoprene molecules.Natural farnesene consists of two isomers β-farnesene and α-farnesene.Because of the conjugated double bond in its molecular structure,β-farnesene has been widely used in new materials,energy,fine chemicals and other fields.In energy field,β-farnesene can be used as lubricating oil and high-performance fuel because of its high cetane number,low temperature performance.In fine chemicals field,β-farnesene can be used as a synthetic precursor of nutritional chemicals and insect repellent.Traditional farnesene sources rely on chemical synthesis and extraction of natural products.Chemical synthesis steps are complex and the conversion is low.The extraction process is difficult to large-scale production because of the raw materials constraints.The development of synthetic biology technology provides a new biosynthesis mode for β-farnesene.The construction of engineering strain producing β-farnesene makes it possible to produce β-farnesene on a large scale by biological fermentation.Saccharomyces cerevisiae is one of the commonly used strains to produce sesquiterpenes including β-farnesene.There have been many reports on the production of β-farnesene by modified Saccharomyces cerevisiae,and the mass production process of β-farnesene has been reported by foreign research institutions.Most of the studies reported in China remain in engineering strain construction or small-scale fermentation stage,and relevant studies are relatively mature.The amplification fermentation process of the modified engineering strain and the separation and extraction process which is suitable for industrial scale have become bottlenecks blocking the industrialization of the production of β-farnesene.Therefore,this study was based on the engineering strain of Saccharomyces cerevisiae producing farnesene constructed by our research group.By optimizing the fermentation conditions and control strategy in the process of fermentation amplification,developing the separation technology in the process of tonnage fermentation,30 tons of β-farnesene production by saccharomyces cerevisiae engineering strain and product separation were realized.The research contents and results are as follows:1.Completed the establishment of 2.5L fed-batch fermentation process and the development of fed-batch control strategy.The composition of synthetic medium suitable for engineering strain was determined by optimizing the medium.By exploring the growth law of the engineering strain,the initial glucose concentration of fermentation 20 g/L,transfer time 12 h,inoculation amount 8～10% and the starting point of feeding is the first complete consumption time of ethanol 34～40 h.Combined with the fermentation characteristics of the engineering strain,a new strategy of dissolved oxygen-controlled feeding was designed based on the traditional strategy of dissolved oxygen-controlled feeding to realize automatic sugar limited feeding,and the optimal carbon source for feeding was determined as glucose.2.The amplification fermentation from 2.5 L-30 L-100 L using engineering Saccharomyces cerevisiae strains was completed.The 100 L scale fermentation level was close to the small test level owing to the amplification effect was eliminated by adjusting the fermentation control strategy.And the separation process in pilot process was developed.The final fermentation yield of 2.5 L was 27.6 g/L,30 L was 24.6 g/L,and 100 L was 11.4 g/L.The optimal separation process of β-farnesene was determined as centrifugal extraction and vacuum distillation.The operation parameters of centrifugal extraction and vacuum distillation were optimized in the following experiment.3.Based on the 100 L pilot fermentation process,the fermentation scheme,feeding scheme and product separation and purification scheme of 30 tonsβ-farnesene amplification fermentation process was designed.The mass fermentation finally yield of 30 tons was 18.0 g/L,and the β-farnesene product with purity of 95% was prepared by disc centrifugation,centrifugal extraction and vacuum distillation. |
