Information on the inserted DNA sequences

The production strain Komagataella phaffii MXY0541 contains inserted DNA sequences introduced through successive genetic modifications. These comprise expression cassettes for soybean leghemoglobin (lgb2), a modified mxr1 transcription factor, and endogenous heme biosynthesis genes from K. phaffii. The soybean lgb2 coding sequence was codon-optimized for expression in K. phaffii. Publicly available information fully supports the structure of the genetic cassettes 1 and 2. The structures of inserts 3 and 4 were reconstructed from the available molecular characterization, whole-genome sequencing, PCR analyses and application documents, as complete information on their organization and sequence composition has not been publicly disclosed. In addition, PCR analysis and whole-genome sequencing demonstrated that the strain MXY0541 contains several copies of a CPG oxidase cassette and additional copies of the codon-optimized soybean lgb2 gene relative to the intermediate strain MXY0291. Detailed information on the organization, regulatory elements, copy number and genomic insertion sites of these additional inserted DNA sequences has not been publicly disclosed. 

(1) The codon-optimized soybean lgb2 gene, encoding leghemoglobin C2 from Glycine max, is regulated by the native AOX1 promoter and AOX1 terminator from K. phaffii. Whole-genome sequencing of the intermediate strain MXY0291 identified sixteen copies of this primary expression cassette. High levels of soybean leghemoglobin expression are expected due to the multiple integrated copies of the AOX1-regulated expression cassette.

(2) The modified mxr1 gene is regulated by the native AOX1 promoter and AOX1 terminator from K. phaffii. Due to the cloning strategy, the encoded Mxr1 protein contains six additional amino acids at its N-terminus compared with the native protein. Mxr1 is a transcriptional activator of the AOX1 promoter, resulting in increased expression of the lgb2 gene, the introduced heme biosynthesis genes, and the mxr1 expression cassette itself.

(3) Insert 3 contains integrated expression cassettes originally derived from transformation vector pMX349. It comprises expression cassettes for the endogenous hem1, hem3, hem14 and hem15 genes of K. phaffii, encoding four enzymes of the conserved heme biosynthesis pathway. The genes are regulated by additional copies of the native AOX1 promoter and were introduced to increase intracellular heme production required for soybean leghemoglobin synthesis. The order of the expression cassettes and the identity of the terminators have not been publicly disclosed.

(4) Insert 4 contains integrated expression cassettes originally derived from transformation vector pMX346. It comprises expression cassettes for the endogenous hem4, hem2, hem12 and hem13 genes of K. phaffii, encoding four enzymes of the conserved heme biosynthesis pathway. The genes are regulated by additional copies of the native AOX1 promoter and were introduced to increase intracellular heme production required for soybean leghemoglobin synthesis. The hem13 expression cassette is truncated due to incomplete integration. The order of the expression cassettes and the identity of the terminators have not been publicly disclosed.

Note

-The strain MXY0541 was developed from the parental strain Bg11 (MXY0051) through a series of sequential genetic modifications, generating the intermediate strains MXY0197, MXY0199, MXY0203, MXY0213, MXY0260 and MXY0291 before the optimized production strain MXY0541 was obtained.
-The intermediate strain MXY0203 was generated by sequential transformation with plasmids pMX349 and pMX346, carrying expression cassettes for the eight endogenous heme biosynthesis genes (hem1, hem2, hem3, hem4, hem12, hem13, hem14 and hem15). Prior to transformation, both plasmids were linearized with the restriction enzyme PmeI, and the expression cassettes were introduced into the recipient strain by electroporation. Antibiotic resistance markers flanked by lox sites were subsequently removed by Cre-lox recombination, resulting in stable chromosomal integration of the expression cassettes under the control of additional copies of the native AOX1 promoter.
-The intermediate strain MXY0260 was generated by co-transformation with a linear pAOX1-MXR1-TAOX1 expression cassette. Transformants were selected using a transient plasmid carrying antibiotic resistance genes, which was subsequently cured and confirmed absent by PCR and antibiotic sensitivity testing.
-The intermediate strain MXY0291 was generated by co-transformation with a linear pAOX1-LGB2-TAOX1 expression cassette. Whole-genome sequencing confirmed that the genome of MXY0291 contains sixteen copies of the pAOX1-lgb2-TAOX1 expression cassette, one mxr1 expression cassette, one partial integration derived from transformation vector pMX349, and two to three partial integrations derived from transformation vector pMX346.These integrated vector-derived sequences contain expression cassettes for the endogenous heme biosynthesis genes but do not contain antibiotic resistance genes or bacterial origins of replication.
-The optimized production strain MXY0541 was generated by introducing additional lgb2 and hem13 sequences to further increase soybean leghemoglobin production. PCR analysis and whole-genome sequencing confirmed the presence of these additional sequences relative to MXY0291. The introduced DNA remained stable over 150–200 generations of growth. Molecular characterization demonstrated the absence of antibiotic resistance genes, functional origins of replication and residual vector backbone sequences in the final production strain. Detailed information on construct design, exact genomic insertion loci, and the complete sequence composition of some inserted DNA sequences has not been made publicly available.