Biosynthesis Of α-farnesene In Pichia Pastoris

Microbial production ofα-farnesene is a promising alternative to traditional petroleum-based processes.Although it has been reported thatα-farnesene can be heterologously produced by conventional model strains such as Escherichia coli and Saccharomyces cerevisiae,its fermentation scale is not easy to achieve high-density fermentation.Pichia pastoris is a good platform for the production of isoprene and has the potential for large-scale production ofα-farnesene at high density.Currently,efforts to improveα-farnesene production in P.pastoris mainly focus on cytoplasmic engineering,whereas comprehensive engineering of multiple subcellular compartments is rarely reported.Here,we proposed dual regulation of cytoplasm and peroxisome to boostα-farnesene synthesis in P.pastoris,thus the resultant strain produced 2.18±0.04 g·L^-1,which was 1.3times and 2.0 times than that of the strains with solely peroxisomal or cytoplasmic engineering,respectively.Theα-farnesene production achieved 2.81±0.05 g·L^-1 in shake flasks after carbon source co-feeding and fermentation optimization.Therefore,we propose these strategies as efficient approaches to enhancingα-farnesene production in P.pastoris,which might open new possibilities for bioproduction of other value-added chemicals.The main research results are as follows:（1）Based on the cytoplasm of P.pastoris,a series of metabolic engineering was carried out to promote the biosynthesis ofα-farnesene.Firstly,construct a phylogenetic tree ofα-farnesene synthase（AFS）,and select 20 kinds of AFSs from different plants to screen the most suitable AFS for the production ofα-farnesene in P.pastoris.Then overexpress the rate-limiting enzymes of the mevalonate（MVA）pathway and theα-farnesene synthesis pathway and increase the supply of acetyl-Co A,and finally obtain a cytoplasmic engineering strain X33-6.The yield ofα-farnesene was 1.07±0.03 g·L^-1.（2）It was proved for the first time that the IUP could be used as a two-step shortcut for the synthesis of isopentenyl pyrophosphate（IPP）and dimethylallyl pyrophosphate（DMAPP）to promote the efficient synthesis ofα-farnesene in the peroxisome of P.pastoris.The isopentenol utilization pathway（IUP）was introduced into the peroxisome of P.pastoris,resulting in the target strain X33-CHKLIPK-e PTS1.Compared with the control strain X33,the IUP-activated strain X33-CHKLIPK-e PTS1 had a significant increase of the IPP+DMAPP library by 6.8 times.On this basis,the introduction of theα-farnesene synthesis pathway and optimization of key enzymes copy number result in peroxisomal engineering strain X33-8 and the titer ofα-farnesene was 1.69±0.06 g·L^-1.Compared with strain X33-AFS_Md-e PTS1,the production ofα-farnesene was significantly increased by 2.2 times,realizing the efficient production ofα-farnesene.（3）Cytoplasm and peroxisome were synergistically modified to boostα-farnesene synthesis in P.pastoris.The resultant strain X33-10 produced 2.18±0.04 g·L^-1,which was 1.3times and 2.0 times than that of the strains with solely peroxisomal（X33-8）or cytoplasmic engineering（X33-6）,respectively.（4）Mixed carbon source cultivation and optimization of fermentation conditions can improve cell growth and increase the production ofα-farnesene.When sorbitol was used as the auxiliary carbon source and the oleic acid fraction was 0.5（C-mol oleic acid:C-mol sorbitol）,the highestα-farnesene yield was 2.51±0.04 g·L^-1.Based on the optimized culture conditions,the finalα-farnesene yield of strain X33-10 was 2.81±0.05 g·L^-1,and the yield was 0.15 g·g^-1.