Heterologous Expression Of Linoleic Acid Isomerase And Product Biosynthesis In Oleaginous Fungus

Conjugated linoleic acid (CLA) is a generic term used to describe a mixture of positionaland geometric isomers of linoleic acid (LA) with conjugated double bonds. In the past threedecades, CLA has attracted much attention because of its biologically beneficial functions. Tomeet the requirements for medicinal and nutritional purposes, CLA isomers need to bebiologically safe and highly specific. Therefore biosynthesis of CLA has become theinevitable technology trends. In this dissertation, the linoleic acid isomerase gene fromPropionibacterium acnes (PAI) was successively expressed in Escherichia coli, Mortierellaalpina and Yarrowia lipolytica. Yarrowia lipolytica was confirmed to be the properbiosynthesis system for trans-10，cis-12-CLA. A recombinant Yarrowia lipolytica strain withability to produce large quantities of trans-10, cis-12-CLA was constructed by geneticmodifications. The trans-10, cis-12-CLA yield was further improved by optimization ofcultivation conditions. The main results were described as follows:(1) Three new recombinant E.coli strains were constructed and used to over-expressthree types of recombinant PAI: PAI,.PAI fused with C-terminal6×his-tag and PAI fusedwith N-terminal6×his-tag. The purified his-tag PAI was used as antigen for the generation ofPAI-specific polyclonal antibodies in rabbits. To analysis the recombinant PAI expression inE.coli, SDS-PAGE and western blot were carried out. PAI activity in recombinant E.coli strainwas confirmed by gas chromatogram. The results showed that, under the same cultivation andinducible expression conditions, three recombinant E.coli strains successfully expressedrecombinant proteins which had linoleic acid isomerase activity. The total recombinant PAIproduction levels were similar among three recombinant E.coli strains. His-tag had anoticeable negative effect on protein solubility and PAI activity. C-terminal his-tag had astronger negative effect on these aspects than N-terminal his-tag. To obtain maximum activePAI, PAI gene should be expressed in oleaginous fungus without any affinity purification tags.(2) The production, germination rate, collection method and antibiotics sensitivity ofspores were investigated. The optimum cultivation condition for spores production is that M.alpina was cultivated on slant PDA medium at28℃for30days. To remove mycelia andminimize spores losses, the spore suspension was successively filtrated by250μm fabric and20μm membrane. Hygromycin B was used as selection marker and the optimumconcentration for transformation was0.8-1.5mg/mL. The recombinant vectors pD4-pai andpBIG-pai were constructed for microprojectile bombardment and Agrobacteriumtumefaciens-mediated transformation (ATMT) respectively. Transformation conditions wereinvestigated and the stable transformants were obtained by ATMT. PAI gene integration intochromosomal DNA and transcription were confirmed by PCR analysis, using genomic DNAand cDNA from stable transformants. The results suggested gene manipolation system for M.alpina was established. However the recombinant PAI was not detected by SDS-PAGE andwestern blot analysis. These results revealed that PAI gene was not expressed in M. alpina.Codon usage differences between PAI gene and M. alpina may be the reason for the failure ofPAI expression. (3) Considering the failure of PAI expression in M. alpina, PAI gene was firstlyoptimized according the condon usage bias in Y. lipolytica. Additionally, the Kozak elementwas added before the first AUG codon to prevent the leaky scanning of the ribosome.Mono-copy expression cassettes of native PAI gene (pai) and optimized gene (opai) wereintegrated into genomic DNA of Y. lipolytica. The recombinant PAI is present in all yeasttransformants carrying opai. By comparison, the level of PAI expressed from the native paiwas extremely low and could not be detected by western blot except for two transformants.The average content of trans-10，cis-12-CLA in transformants carrying opai was2.1mg/L,which corresponds to a10-fold increase compared with the transformants carrying pai. Theseresults suggested that codon optimization was essential for heterologous expression ineukaryotes. The expression level of PAI and trans-10，cis-12-CLA production were furtherpromoted with a multiple-copy expression of a codon-optimized gene. The average content oftrans-10，cis-12-CLA in multiple-copy transformants carrying opai was14.3mg/L, whichcorresponds to a6.8-fold increase compared with the mono-copy transformants carrying opai.(4) The genetic modifications of Y. lipolytica were performed to enhance the level ofsubstrate LA and PAI expression. The mono-copy integrative strain with the codon-optimizedgene opai was used as a control strain. To increase PAI substrate (LA) content, the delta-12desaturase gene was cloned from M. alpina and expressed in Y. lipolytica. As a result, PAIcontent in total fatty acids was increased from27.8%to62.2%and trans-10，cis-12-CLAproduction was increased from2.3mg/L to3.8mg/L. To enhance opai expression, thepromoter, hp4d, was replaced by a modified promoter hp16d located upstream of opai. Theexpression level of opai under hp16d was3-fold higher than that in the control strain and thetrans-10，cis-12-CLA production was increased to7.0mg/L. Furthermore, the synergisticeffect of d12expression and enhanced promoter resulted in a4.8-fold increased yield of CLA(11.0mg/L) compared to the control. The multi-copy integration also improved CLA yield:the trans-10，cis-12-CLA yield was increased to36.8mg/L, which corresponds to a total of16-fold increase. The highest CLA yield obtained by the best performing transformantPolh-pINA1292-spoPAI-D12-16was44.9mg/L, corresponding to9.8%in total fatty acids.(5) The non-conventional dimorphic yeast Y. lipolytica has been efficiently cultivated onvarious hydrophobic substances such as alkanes or lipids to produce many extra-orintra-cellular metabolites of industrial significance. Considering the specific properties of Y.lipolytica and the limiting factors for CLA production, YNBDL6medium supplemented withLA was used to increase PAI substrate. At72h of cultivation in flask, the trans-10，cis-12-CLA yield in Polh-pINA1292-spoPAI-D12-16was maximum, which was6.7-foldhigher than that in YPD medium, corresponding to0.3g/L. Interestingly,0.1g/L CLA thereinwas detected in supernatant medium. Increase of nitrogen concentration in medium inhibitedacid production and promoted cell growth, lipid intake and PAI expression. In high nitrogenmedium YN20BDL6, the maximal CLA yield was2.6g/L observed at72h of cultivationwhich was8.7-fold than that in low nitrogen medium. The effect of different LAconcentration on growth characters was investigated. High LA concentration had no negativeeffect on Y. lipolytica growth and lipid accumulation. Moreover, the trans-10，cis-12-CLA yield increased according with LA concentration. The CLA production were separately0.9g/L,2.6g/L and3.7g/L in medium added with33%,65%and96%LA. The overall modifiedstrain was cultivated with YN20BDS medium supplemented with soybean oil which is wildlyavailable, low-cost and rich in LA. The growth characters, lipid accumulation and CLAproduction in YN20BDS were similar with those in the medium supplemented with65%LA.The maximum CLA production in cells was2.5g/L. Polh-pINA1292-spoPAI-D12-16wascultured in5-L fermentor with YN20BDS medium. The maximum CLA production in cellswas3.1g/L and observed at the late stage of rapid growth (38.5h of cultivation). A largeportion of CLA (0.9g/L) was detected in supernatant medium simultaneously.The total CLAproduction was4g/L, corresponding to90-fold higher than that in YPD medium and20-foldhigher than the highest production of trans-10, cis-12-CLA by biosynthesis. The extracellularactivity of PAI in medium at24h of cultivation was tested and optical microscopic analysiswas performed to evaluate the cell integrity. As a result, there was no activity of PAI to bedetected in supernatant culture and no cell fragmentation was observed. These resultssuggested that the CLA in culture supernatant was not due to PAI secretion and cell lysis. Thepossible explanation was the result of fatty acid exchange between intracellular andextracellular matrixes.