Metabolic Engineering Of Pichia Pastoris For Efficient Synthesis Of Porcine Myoglobin And Soybean Hemoglobin

Porcine myoglobin（P-Mb）and soybean hemoglobin（S-Hb）are single-subunit proteins comprising a globin chain and a heme cofactor.They hold broad prospects for application in various fields,such as artificial meat development,dietary supplements,and artificial metalloenzymes.Pichia pastoris（syn.Komagataella phaffii）is considered the preferred choice for synthesizing hemoproteins due to the advantages of secretory expression and high-cell-density fermentation.However,there are still some bottlenecks that hinder the efficient synthesis of high-active hemoproteins in Pichia pastoris.The first bottleneck is the low expressional level and severe degradation of the globin component in multitudinous hemoproteins.The second one is the spatial isolation of heme biosynthesis.Heme intermediates must cross the inner and outer mitochondrial membranes several times during heme biosynthesis,reducing the efficiency of heme synthesis and utilization.The third one is the inadequate heme supply resulting from the rate-limiting steps during heme biosynthesis.The fourth one is the imbalance in the distribution of metabolic flow between heme biosynthesis and globin expression.To solve these issues,this study constructed an efficient heme-supply P.pastoris chassis by selecting an appropriate expression strategy for globin,removing the spatial segregation during heme biosynthesis,optimizing precursor synthesis,assembling rate-limiting enzymes using protein scaffolds,and inhibiting heme degradation.Based on the robust chassis,the heme-responsive biosensor was constructed to finely balance heme biosynthesis and globin expression,thereby enabling the efficient synthesis of high-active P-Mb and S-Hb.The main results in this study are as follows.（1）Construction and optimization of a secreted expression system for globin.By investigating the impact of various P.pastoris hosts,promoter elements,gene copy numbers,and other conditions on the expression of the globin component in hemoprotein,an appropriate expressional strategy for globin in P.pastoris was chosen,which included P.pastoris X33,methanol-inducible expression system based on P_AOX1,and single-copy integrated expression for the globin gene.Subsequently,the transcription of globin was enhanced,and its degradation was inhibited by overexpressing the P_AOX1transcriptional activators（Mit1,Mxr1,and Prm1）and knocking out proteases（Yps1-1,Prb1,and Pep4）.Finally,in the optimal platform strain for globin expression(X33-Δku70-Δyps1-P_GAP-Mit1,namely as P1),the P-Mb titer reached 162.46±11.12 mg L^-1,which was 21 times higher than the reported value of 7.73 mg L^-1.The S-Hb titer increased to 184.35±17.14 mg L^-1,representing a 22.06%improvement compared to the previous titer of 151.03 mg L^-1.（2）Reconstruction of the heme biosynthetic pathway.Through bioinformatics analysis and fusion expression of heme biosynthetic enzymes with fluorescent protein,the subcellular localization of heme biosynthetic enzymes was first revealed in P.pastoris.Five heme biosynthetic enzymes（Hem2p,Hem3p,Hem4p,Hem12p,and Hem13p）were present in the cytoplasm,while the other three enzymes（Hem1p,Hem14p,and Hem15p）were localized in the mitochondria.The MLS for Hem1p,Hem14p,and Hem15p were identified,including MLS_Hem1p located in the first 52 residues in the N-terminal,while MLS_Hem14p and MLS_Hem15pboth located between the 61 and 70 residues in the N-terminal.Then,the transfer of 5-aminolevulinic acid（ALA）synthesis from mitochondria to the cytoplasm was performed by removing the MLS from Hem1p or introducing alternative ALA biosynthetic enzymes from bacteria.The results showed that the titers of ALA in these engineered strains were extremely low.Therefore,the synthesis of ALA in the mitochondria should be maintained during the reconstruction of the heme biosynthetic pathway.Subsequently,the titer of ALA was increased to 100.09±1.29 mg L^-1 by overexpressing the native HEM1 gene(P_GAP-HEM1).Finally,the functional expression of the mitochondrial enzymes Hem14p and Hem15p was assessed in the cytoplasm.The results showed that the titer of heme increased by 41.94%by co-expressing the MLS-truncated versions of Hem14p and Hem15p,HEM14_71-561and HEM15_71-375.（3）Enhancement of the rate-limiting steps during heme biosynthesis.Enhancements to the key steps in the reconstructed heme biosynthetic pathway,such as assembly of the rate-limiting enzymes Hem2p,Hem3p,and Hem4p using protein scaffolds（90.24%increase in heme titer）,enhancement of the expression of HEM13 by replacing its native promoter with the strong constitutive promoter P_GAP to alleviate the accumulation of the intermediate coproporphyrinogen III（83.45%increase in heme titer）,and knockout of the heme oxygenase Hmx1p（1.66-fold increase in heme titer）,further improving heme accumulation.The enhanced heme-supplying strain HEME-9 was generated by integrating these engineering strategies for the heme biosynthetic pathway,which achieved a heme titer of 2.93±0.27 mg L^-1,a 37.58-fold increase compared to the original strain X33-Δku70.Finally,the strategies employed in the P1 strain were applied in the heme-supplying strain HEME-9,generating the final production strain P1H9.Due to the sufficient intracellular availability of heme,the titer of P-Mb and S-Hb increased by 52.01%and 55.43%,respectively,corresponding up to 246.95±19.46 mg L^-1 and286.53±14.29 mg L^-1.Moreover,the heme-binding ratios of P-Mb and S-Hb increased by2.87-fold and 3.67-fold,respectively,corresponding reached 60.02±6.98%and 79.47±6.72%.In addition,the specific peroxidase activities of P-Mb and S-Hb increased by 38.10%and29.31%,respectively.（4）Fine-tuning heme synthesis to balance the expression of globin.Endogenous genes with significant differences in transcriptional levels were identified through comparative transcriptomics analysis with and without heme addition.Next,the promoters of these genes,along with promoters previously reported in the literature to be responsive to heme,were used to express the reporter gene lac Z,respectively.The two sensitive positive regulatory elements in response to heme were obtained(P_2-10235and P₃₀₅₁₆).Subsequently,two positive regulators,CRISPR/d Cas9,three degradation tags on the d Cas9 protein,and two g RNAs were used to design various heme-responsive biosensors for dynamically controlling the expression of m Scarlet.In the conditions using the optimal regulatory elements,including P_2-10235,K3K15,and g RNA2_{m Sca},the dynamic regulation of m Scarlet expression occurred at 22-36 h and 36-48h,with periods of 14 h and 12 h,respectively.In the following,the optimized heme-responsive biosensor was applied to fine-tune the expression of the key gene for heme biosynthesis（HEM2）in the hemoprotein-producing strain P1H9.In the final strain P1H9-R3,the dynamic regulation of heme biosynthesis occurred at 18-28 h,28-40 h,and 40-48 h,with periods of 10 h,12 h,and8 h,respectively.Finally,the functional expression of P-Mb and S-Hb was verified in the P1H9-R3 strain.The results showed that the enhanced level of heme supply was fine-tuned,resulting in a 20.12%and 30.02%increase in the titers of P-Mb and S-Hb,as well as a 16.13%and 18.22%improvement in the specific peroxidase activities of P-Mb and S-Hb,respectively.Furthermore,the heme-binding ratios of P-Mb and S-Hb reached 66.33±5.20%and 88.90±6.48%,respectively.