Screening Of Lipase Producing Fungus,gene Cloning,codon Optimization And Expression In Pichia Pastoris

The Galactomyces geotrichum lipase（GGL）shows a remarkably high selectivity towards cis-9 unsaturated substrates with long fatty acyl chains.It is commonly used in detergent,food,feedstuff and oleochemistry.However,the expression level of GGL is relatively low in natural conditions,which limits its application in industry.Thus,it is of great significance to realize its high-level expression in heterologous host using genetic engineering technique and fermentation optimization strategy.1.A lipase producing strain with high-activity was isolated from oily soil or water samples.A 344 bp internal transcribed spacer（ITS-rDNA）sequence was amplified using universal primers ITS.Blast showed the sequence was 99%identical to Galactomyces geotrichum J8M-14（JN227034）.Combined with morphological,physiological and biochemica characteristics,the strain was identified as Galactomyces geotrichum,named G.geotrichum FZ-4.The lipase from G.geotrichum FZ-4 showed optimum activity at pH 7.5and 40℃.The lipase retained more than 50%residual activity after incubation for 30 min at pH 7.0-9.0 and below 45℃.The lipase activity was significantly stimulated by Ca²⁺and Mn²⁺,and slightly inhibited by Co²⁺and Na⁺,while significantly inhibited by Fe²⁺,Fe³⁺and Zn²⁺.The enzyme showed preference for p-NPP（C16）.2.With alignment of nucleotide sequence of Galactomyces sp.lipase gene in NCBI,the conservative nucleotide sequence was obtained.The lipase gene of G.geotrichum FZ-4（No.KP143749）was cloned using cDNA and genomic DNA as template,while the degenerate primers were designed based on the conservative nucleotide sequence of lipases.Analysis of nucleotide sequence revealed that the ORF of GGL I has 1692 bp,encoding563 amino acid residues including a potential signal sequence of 19 amino acid residues.The molecular weight of mature peptide is 60.44 kDa and its theoretical pI is 5.39.The sequence aligment with Blast showed that the CDS of G.geotrichum FZ-4 was 98%identical to G.candidum BT107 for lipase I（No.ABD67166）.The GGL I was hydrophilic amino acid and localized in extracellular of the strain,mitochondrion and ribosome with no transmembrane structure.The GGL I belongs to transferase with transport and binding function.The secondary structure was mainly made up ofα-helix and random coil.The tertiary structure of GGL I showed that it was presented as monomer with a flap above the active site.3.Analysis using a graphical codon usage analyser revealed that the GGL I-wt harbours nearly 9%codons,which represented<10%usage in P.pichia.The hydr olytic activity of GGL I-op toward olive oil from the supernatant was 150 U/mL,w hile GGL I-wt had no activity due to the low-usage codons.There was 7.8%less GC percentage content in the GGL I-op than in the GGL I-wt.The CAI,which rep resents the predicted expression level of a gene,was upgraded from 0.72 to 0.85 af ter codon replacement.Six recombinants possessing GGL I named X33/Zα-ggl1-op,SMD1168/Zα-ggl1-op,GS115/9K-ggl1-op,SMD1168/9K-ggl1-op,X33/GAP-ggl1-op a nd SMD1168/GAP-ggl1-op were constructed.A clone named X33/Zα-ggl1-op-8 was obtained and its initial lipase activity was 150 U/m L.The culture supernatants（300mL）were ultrafiltrated through a 10 k Da membrane,yielding a concentrated lipase solution with a 84%yield and a 1.26-fold increased in specific activity,519.69 U/mg.After affinity chromatography on a Ni-NTA column,aspecific lipase activity of1154.64 U/mg with a 62.22%yield and 2.8-fold increased was obtained.4.Real-time PCR was applied to determined the copy number of GGL I gene in target clones.The GGL I gene copy number were covered from 1 to 5.The enzyme activity was53,121,147,106 and 45 U/mL respectively,when GGL I gene copy number from 1 to 5.The enzyme activity reached maximum when GGL I gene copy number was 3.5.The maximum GGL I lipase activity was obtained with a 2%inoculum size and the optimal culture condition was achieved by the daily addition of 1.5%（v/v）methanol to a250 mL shaking flask containing 35 mL culture medium for 96 h at pH 7.0 and 25℃.The purified GGL I exhibited optimum lipolytic activity at pH 8.0 and 35℃.The enzyme retained stable at pH 7-9 and temperature below 45℃.The enzyme activity was stimulated by Mg²⁺,Ca²⁺,Mn²⁺and Cu²⁺,and inhbited by Fe²⁺,Fe³⁺,Zn²⁺and Co²⁺.The enzyme showed preference for p-NPO（C8）.Target protein of 63 kDa was identified as GGL I by mass spectrometry.After EndoH digestion,the molecular weight of GGL I decreased to 60kDa,much similar to the calculated GGL I.Thus,it was demonstrated that GGL I had been glycosylated in protein processing.