Abstract
Screening for a new yeast as an alternative host is expected to solve the limitations in the present yeast expression system. A yeast sample which was isolated from the traditional food starter ‘ragi’ from Malaysia was identified to contain Meyerozyma guilliermondii strain SMB. This yeast-like fungus strain SMB was characterized to assess its suitability as an expression host. Lipase activity was absent in this host (when assayed at 30 °C and 70 °C) and Hygromycin B (50 μg/mL) was found to be its best selection marker. Then, the hyg gene (Hygromycin B) was used to replace the sh ble gene (Zeocin) expression cassette in a Komagataella phaffii expression vector (designated as pFLDhα). A gene encoding the mature thermostable lipase from Bacillus sp. L2 was cloned into pFLDhα, followed by transformation into strain SMB. The optimal expression of L2 lipase was achieved using YPTM (Yeast Extract-Peptone-Tryptic-Methanol) medium after 48 h with 0.5% (v/v) methanol induction, which was 3 times faster than another K. phaffii expression system. In conclusion, a new host-vector system was established as a platform to express L2 lipase under the regulation of PFLD1. It could also be promising to express other recombinant proteins without inducers.
Highlights
Komagataella phaffii, which was previously known as Pichia pastoris, is the most common host for recombinant protein expression in industries
Local yeast isolate collected from a traditional fermented food starter in Malaysia was cultured in YPD medium [Yeast Peptone Dextrose: 1% (w/v) yeast extract (Becton Dickinson, Franklin Lakes, NJ, USA), 2% (w/v) peptone (Becton Dickinson, Franklin Lakes, NJ, USA), 2% (w/v) agar (Becton Dickinson, Franklin Lakes, NJ, USA), and 2% (w/v) dextrose (Chemiz, MY)]
The Basic Local Alignment Search Tool (BLAST) results showed that the the seseqquueennccee wwaass iiddeenntitciaclaltotoMM. g.ugiluliielrlmieromndoiin. dFioi.r Fyoearrys,etahres,htehteerohtehtaelrlioctahnadlliscpaonrodgesnpooursogyeeansot,uMs .yeast, M. gugiulliilleiremrmoonnddiiii, hhaass bbeeeenn uusseeddiinnrribibooflflaavvininprpordoudcuticotnio. nT.hTe heevoelvuotilountaiorynarreylatrieolnastihoipnsshoifpsstroafinstSrMainB SMB usingusriDngNrADNITAS IrTeSgiroegnioanrearileluilslutrsatrtaetdedininFFigiguurree11
Summary
Komagataella phaffii, which was previously known as Pichia pastoris, is the most common host for recombinant protein expression in industries. Most foreign genes are expressed under the control of the K. phaffii alcohol oxidase 1 promoter (PAOX1), where methanol is a vital inducer. Some recombinant proteins require a longer time for optimal production and high methanol induction when expressed under the regulation of PAOX1 [1]. The negative effects of excessive methanol usage in this system have not been discussed much, since the recombinant proteins will normally be purified before use. Direct consumption of crude proteins is unsafe for human and feed industries. It is important to have a new yeast expression system from food sources as an alternative host with minimal enzymes production time to reduce the production cost and minimize the methanol toxicity effects
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