Study Of Fermentation Bioprocess For Lycopene Biosynthesis By Escherichia Coli DH416 And A Kinetic Model

Lycopene being an important kind of carotenoid with a high economic value,has been widely used in feed,food,health care products,cosmetics,medicine and other industries.Generally,there are many ways to produce lycopene,but the lycopene biosynthesis by microbial fermentation has the advantage of high yield with the low cost and little harm to environment.Therefore,it has the potential of industrialization at large scale production.Previosuly,a large number of studies have shown that the introduction of metabolic pathway of mevalonate(MVA)into microbial cells can effectively improve the production level of lycopene,but the production and yield of lycopene is still very low.Therefore,in this study,we select E.coli DH416 as the research strain,and carry out the optimization of fermentation process in a 5 L bioreactor.Moreover,we the established the fermentation kinetics model and studied the lpxm gene for improving the growth of the strain,in order to achieve the high-efficiency expression of lycopene and cell growth of the recombinant strain.The results of this study are as follows:(1)Optimization of lycopene fermentation by Escherichia coli DH416 in a 5 L bioreactorThe optimal conditions of lycopene production in the test tube culture were determined as follows:glucose concentration of 10 g/L,yeast powder concentration of 2.0 g/L,inducer L-arabinose concentration of 1.0%(w/v),inducer temperature of 30℃.Based on the test tube culture conditions,the fermentation conditions of engineering strain DH416 were optimized in a 5 L bioreactor.After 9 h fermentation,glucose was added to keep the growth of bacteria,which helps in regulating the physiological parameters of cell metabolism,such as the rate of carbon dioxide release(CER)and the rate of oxygen consumption per unit volume(OUR).Moreover,ammonia solution regulated the pH,and provided nitrogen source for the growth of cells,which increased the lycopene production by 19 times(1480.16 mg/L vs 76.23 mg/L).Furthermore,the ability of lycopene production per unit cell was also increased by 4.1 times.The results showed that recombinant E.coli DH416 has the potential of producing lycopene.(2)Study on the kinetic model of lycopene production by engineered Escherichia coli DH416The fermentation process of engineered Escherichia coli DH416 strain concluded the importance of double substrate culture,in which glucose is the main carbon source and yeast powder is the main nitrogen source.Therefore,it is necessary to establish a double substrate limited fermentation kinetic model to describe the fermentation process.Using batch culture in a 5 L bioreactor,combined with the macro metabolic physiological parameters of lycopene engineering Escherichia coli,the two substrate limited fermentation kinetic model was established by using the Monod kinetic model,Herbert Pirt equation and Luedeking Piret equation.In this kinetic model study,we used the H-P equation to express the relationship between the consumption of glucose as carbon source and the growth of bacteria.Moreover,L-P equation was sued to describe the relationship between lycopene synthesis and the growth of bacteria in the exponential growth period,whereas the bergter model was used to establish the relationship between nitrogen source and cell growth.Ultimately,the fourth-order Runge Kutta method was used to calculate the parameters of the above mentioned relationships.Then,the fitting curve of the specific growth rate,the specific substrate consumption rate,the specific production rate and the specific oxygen consumption rate with time in the fermentation process,were compared with the experimental curve.The trend was matched in general.It was proved that the double substrate restriction model can describe the growth and fermentation process of bacteria,which could be used as a great guiding significance for fermentation process development.(3)Physiological function of lpxm gene in lycopene producing strain DH416In the fermentation of strain DH416 with a high lycopene production and strain DH411 with a low lycopene production,we found that under the condition of self induction,it was difficult to obtain the large cell mass and the high lycopene production level at the same time.Considering that there might be a competition between the cell growth and lycopene production.One of the differences between DH411 and DH416 is that DH416 integrated the expression element GC to lpxm site.Through the construction of lpxm gene multi-copy plasmid pET3b-lpxm,the fermentation results showed that the cell growth increased by 3.5 times after over expression of lpxm gene.The results showed that the growth of cells increased by 4 times and the production of lycopene decreased by 13 times,which indicated that overexpression of lpxm gene could effectively promote the growth of E.coli DH416,and also proved as the competitive relationship between cell growth and lycopene production.(4)Based on the integration method of Escherichia coli producing lycopene,an integrated engineering strain for pinene production was constructed.Finally,the downstream pathway of MVA was integrated into the lpxm site of DH411.The original lycopene synthesis pathway in nonessential 23 region was knocked out,and the pinene synthesis pathway in nonessential 8 region was integrated.As a result,the yield of the engineered strain reached to 50±0.5 mg/L.In summary,this method can provide basis for the production of terpenoids with isoprene pyrophosphate(IPP)and dimethylallylpyrophosphate(dmapp)as precursors.