MXY0541 Pichia pastoris (new name: Komagataella phaffii) is a genetically modified strain of the parental strain Bg11 (MXY0051). The production strain MXY0541 is an optimized version of strain MXY0291.
MXY0541 was modified to express the leghemoglobin gene from soybean (CS-lgb2-SOYBN), which was codon-optimized for P. pastoris and expressed under the strong native alcohol oxidase 1 promoter (P-aox1-PICPA).
Like MXY0291, the optimized strain MXY0541 contains extra copies of 8 native P. pastoris heme biosynthetic enzymes driven by the P-aox1-PICPA promoter to upregulate heme biosynthesis, increasing intracellular concentration of heme to generate sufficient heme-bound soy leghemoglobin. A modified transcription factor mxr1 gene (CS-mxr1-PICPA) was expressed under the P-aox1-PICPA promoter. The Mxr1 protein activates the P-aox1-PICPA promoter leading to increased expression of P-aox1-PICPA-driven CS-lgb2-SOYBN, heme biosynthesis genes, and CS-mxr1-PICPA itself.
The genome of the original production strain MXY0291 has been completely sequenced and confirmed to contain the following sequences:
- 16 copies of P-aox1-PICPA-CS-lgb2-SOYBN-T-aox1-PICPA
- 1 copy of P-aox1-PICPA-CS-mxr1-PICPA-T-aox1-PICPA (please note conflicting information about the utilized terminator in original strain MX0291 (GRAS notice – T-fdh1-PICPA; FSANZ application – T-aox1-PICPA))
- 1 copy of a portion of transformation vector pMX349 - containing genes encoding heme biosynthesis enzymes 1 (CS-hem1-PICPA), 3 (CS-hem3-PICPA), 7 (CS-hem14-PICPA), and 8 (CS-hem15-PICPA) (no antibiotic resistance gene, no origin of replication)
- 2-3 copies of a portion of transformation vector pMX346 - containing genes encoding heme biosynthesis enzymes 4 (CS-hem4-PICPA), 2 (CS-hem2-PICPA), 5 (CS-hem12-PICPA), and 6 (CS-hem13-PICPA) (no antibiotic resistance gene, no origin of replication)
MXY0541 contains the following additional sequences, confirmed by PCR and whole genome sequencing:
- several copies of a CPG oxidase gene cassette (CS-hem13-PICPA), which was truncated in Mx0291
- additional copies of the lgb2 gene (CS-lgb2-SOYBN)
The developer states that sequences are stably integrated and present in the strain over 150-200 generations of growth, which was confirmed by PCR. The final production strain does not contain any antibiotic resistance genes.
The purpose of the genetic modification is to obtain soy leghemoglobin by fermentation.
Following fermentation, cells are lysed and physically separated to remove insoluble materials and to concentrate soy leghemoglobin. The resulting liquid concentrate (LegH Prep) is a mixture containing soy leghemoglobin (9% w/w), residual P. pastoris proteins, and added stabilizers. The final product is heat treated to ensure the absence of viable production organism. Small amounts of residual P. pastoris DNA (about 300 mg/L) may also be present in the LegH Prep.
Impossible Foods developed soy leghemoglobin extracts (LegH Prep) as a heme-containing ingredient in meat-like products which imparts a meat-like flavor onto plant based food products. The first product containing LegH from P. pastoris to be commercialized in Australia, New Zealand, the U.S., Canada, Singapore, and Hong Kong was the Impossible Burger.
Sources:
EFSA document
FSANZ document
Patent US20170349906A1