Biosynthesis Of Pinene In Rhodobacter Sphaeroides

Pinene,an important monoterpenoid compound,can be used to synthesize fine chemicals,materials and high-density fuels,thus providing a great potential of being used in biomedical,bio-energy and agricultural fields.Currently,the main approaches for its industrial production is tree-tapping(gum turpentine)or as a byproduct of paper pulping(crude sulfate turpentine,CST).However,the extraction of this molecule from plants is energy consuming,inefficient and taking up substantial expenditure of natural resources because of its low content in pine.Thus,the synthesis of terpenoids in microorganisms becomes a research focus in the field of metabolic engineering and synthetic biology recently.The synthesis of pinene in microorganisms has also been drawed more attention.Currently,the most commonly used chassis are E.coli and Saccharomyces cerevisiae,which are heterotrophic microorganisms,making them costly in large-scale production.At the meanwhile,photosynthetic bacteria Rhodobacter sphaeroides has many kinds of growth and metabolism patterns,and has strong vitality.It can synthesize a large amount of terpnoids by itself and has unique advantages.However,there are few studies on the biosynthesis of pinene by Rhodobacter sphaeroides 2.4.1,the ability of such chassis on pinene production also remains unknown.This paper demonstrated a new approach on the biosynthesis of pinene using photosynthetic bacteria Rhodobacter sphaeroides as chassis.New ways of increasing the yield was also demonstrated.In this paper,two constitutive expression vectors were constructed with two different intensities of promoter: BBa_J95022 and BBa_J95025,based on the wide host plasmids pBBR1mcs-5 and pBBR1mcs-2.Expression strains pBBR5-J22-Gppsps / R.sphaeroides 2.4.1 and PBBR5-J25-Gppsps / R.sphaeroides 2.4.1 was used as host to achieve the biosynthesis of pinene in photosynthetic bacteria.In addition,the expression vectors carrying His-tag was constructed and was used to validate the expression of the target protein.The expression levels of GPPSPS were analyzed by real-time quantitative PCR(qPCR)and Western blot.The results of qPCR showed that BBa_J95025 was a stronger promoter than BBa_J95022.The strains of pBBR5-J22-Gppsps / R.sphaeroides 2.4.1 and pBBR5-J25-Gppsps / R.sphaeroides 2.4.1 were cultured under light-fermentation and shake-flask fermentation respectively.For the former group,the yield of ?-pinene was about 5?g /L,the yield of ?-pinene is 6-10?g / L;for the latter group,the yield of ?-pinene is about 16?g / L and the yield of ?-pinene is about 23?g / L.Thus implied that a strong promoter had a positive effect on pinene production.In order to further increase the production of pinene,two inducible expression vectors were constructed using the strong light-inducible promoter(puc)and a strong chemical-induced promoter(trc).Expression strains pBBR5-puc-Gppsps / R Sphaeroides 2.4.1 and pBBR5-trc-Gppsps / R.sphaeroides 2.4.1,were cultured under light-fermentation and light/shake flask fermentation respectively.The outcome pinene yield of former group was similar to the strain of pBBR5-J25-Gppsps / R.sphaeroides 2.4.1,while the latter group obtained a yield of 25?g/L ?-pinene and 35?g/L ?-pinene under shake flask culturing,and a yield of 60?g/L ?-pinene and 100?g/L ?-pinene under light condition,which was 10-15 times higher than the yield through BBa_J95022 promoter.This work successfully demonstrated the biosynthesis of pinene in Rhodobacter sphaeroides.And reported the production of pinene in Rhodobacter sphaeroides for the first time,This work also compared the production of pinene under different culture conditions was compared,with the highest yield of about 160?g / mL,which was twice as high as the highest yield(~ 80?g/L)obtained from cyanobacteria in previous researches.This paper also established a new method for biosynthesis of pinene in Rhodobacter sphaeroides,and the methods for qualitative and quantitative detection of pinene.