Biosynthesis of High-Active Hemoproteins by the Efficient Heme-Supply Pichia Pastoris Chassis

Abstract

Microbial synthesis of valuable hemoproteins has become a popular research topic, and Pichia pastoris is a versatile platform for the industrial production of recombinant proteins. However, the inadequate supply of heme limits the synthesis of high-active hemoproteins. Here a strategy for enhancing intracellular heme biosynthesis to improve the titers and functional activities of hemoproteins is reported. After selecting a suitable expressional strategy for globins, the efficient heme-supply P. pastoris chassis is established by removing the spatial segregation during heme biosynthesis, optimizing precursor synthesis, assembling rate-limiting enzymes using protein scaffolds, and inhibiting heme degradation. This robust chassis produces several highly active hemoproteins, including porcine myoglobin, soy hemoglobin, Vitreoscilla hemoglobin, and P450-BM3, which can be used in the development of artificial meat, high-cell-density fermentation, and whole-cell catalytic synthesis of high-value-added compounds. Furthermore, the engineered chassis strain has great potential for producing and applying other hemoproteins with high activities in various fields.

Keywords: Pichia pastoris; artificial meat; heme-supply chassis; high-cell-density fermentation; highly active hemoproteins; whole-cell biocatalysts.

Figure 1

Engineering strategies for efficiently synthesizing highly active hemoproteins through optimizing globin expression and moderately enhancing heme supply. a) Optimization of globin expression by overexpressing P _AOX1 transcriptional activators (Mit1, Mxr1, and Prm1) and chaperones (PDI and KAR2), and knocking out proteases (pep4, prb1, and yps1‐1). b) Reconstruction of the heme biosynthetic pathway in the cytoplasm by co‐expressing MLS‐truncated versions of Hem14p and Hem15p (Hem14p_71‐561aa and Hem15p_71‐375aa). c) Enhancement of key steps during heme biosynthesis by overexpressing HEM1 and HEM13, assembling Hem2p, Hem3p, and Hem4p, and deleting heme oxygenase HMX1. Protein scaffolds harboring interaction domains specifically accumulate pathway enzymes tagged with their cognate peptide ligands.^{[ 39 ]} d) Biochemical properties and potential applications of several hemoproteins synthesized in the efficient heme‐supply chassis of P. pastoris. This includes the common characteristic absorption peak and heme‐binding ratio, the specific peroxidase activity of P‐Mb and S‐Hb, the oxygen‐binding property of V‐Hb, and the whole‐cell catalytic efficiency of P450‐BM3. The 3D structure of P‐Mb, S‐Hb, V‐Hb, and P450‐BM3 was obtained from the PDB database (1MWD, 1BIN, 2VHB, and 1FAG, respectively). Abbreviations: HP, hemoprotein; IV, chromosome IV; aa, amino acid; Suc‐CoA, succinyl‐CoA; Gly, glycine; Hem1p, ALA synthase; ALA, 5‐aminolevulinic acid; Hem2p, porphobilinogen synthase; PBG, porphobilinogen; Hem3p, porphobilinogen deaminase; HMB, hydroxymethylbilane; Hem4p, uroporphyrinogen‐III synthase; UPG III, uroporphyrinogen‐III; Hem12p, uroporphyrinogen‐III decarboxylase; CPG III, coproporphyrinogen‐III; Hem13p, coproporphyrinogen‐III oxidase; PPG IX, protoporphyrinogen‐IX; Hem14p, protoporphyrinogen oxidase; PP IX, protoporphyrin‐IX; Hem15p, ferrochelatase; CO, carbon monoxide; BV, biliverdin; Lig, ligand; Dom, domain; ICS, intracellular synthesis; SE, secretory expression.

Figure 2

The optimal expressional platform for the globin component of hemoproteins. a) Selecting a suitable expressional system for P‐Mb globin. SDS‐PAGE analysis is presented in Figure S1a (Supporting Information). The three boxes with brown indicate the control groups for optimizing the P. pastoris host, gene dosage, and fermentation time, respectively. b) Cascade regulation of Mxr1, Prm1, and Mit1 activates the P _AOX1 promoter.^{[ 30 ]} Glucose inhibits P _AOX1 through cytoplasmic Mxr1. Methanol triggers the nuclear translocation of Mxr1, leading to the derepression of P _AOX1 . Prm1 responds to methanol and induces its own expression and that of Mit1, which activates the transcription of the P _AOX1 ‐driven genes. Mit1 feedback inhibits Prm1. GOI, gene of interest. c) Changes in P‐Mb titer after overexpressing chaperones and P _AOX1 transcriptional activators. d) Changes in P‐Mb titer after deleting proteases. CK c,d) indicates the X33‐Δku70 strain harboring the P‐Mb gene. SDS‐PAGE analyses c,d) are presented in Figure S1b (Supporting Information). e) The secretory expression of P‐Mb and S‐Hb and intracellular synthesis of V‐Hb and BM3_mut in the P1 strain. The molecular weight of P‐Mb, S‐Hb, V‐Hb, and BM3_mut containing 6 × His tags predicted by ExPASy were 17.91, 16.35, 16.60, and 118.76 KDa, respectively. Among them, V‐Hb is a homodimer composed of two identical subunits and two heme molecules, thus its molecular weight is 33.20 KDa. CK stands for the cell lysate of the X33‐Δku70 strain without the hemoprotein gene, and M stands for protein ladder. Data presented as mean values ± SD from three independent biological replicates (n = 3). Statistical evaluation (p‐value) compared to the control was conducted by a two‐tailed t‐test. *p < 0.05, **p < 0.01, ***p < 0.001 and NS representing non‐significance (p ≥ 0.05).

Figure 3

Reconstruction of the heme biosynthesis pathway in the cytoplasm. a) Schematic overview of validating the subcellular localization of HBS and their potential MLS. HBSs stands for HBS and their truncated mutants. b) Intracellular localization of native HBS by fluorescence microscopy analysis. CK indicates the recombinant strains of HBS fused with m‐Scarlet observed by white light in the same field of view. The scale bars represent 2 µm. c) Subcellular localization of N‐terminal truncated versions of Hem1p, Hem14p, and Hem15p. d) Effect of cytoplasmic co‐expression of HEM14_71‐561 and HEM15_71‐375 on cell growth and heme synthesis. CK represents the control strain X33‐Δku70. Data presented as mean values ± SD from three independent biological replicates (n = 3). Statistical evaluation (p‐value) compared to the control strain was conducted by a two‐tailed t‐test. *p < 0.05, **p < 0.01, ***p < 0.001 and NS representing non‐significance (p ≥ 0.05). Abbreviations: mSca, m‐Scarlet; MTG, Mitotracker Green FM; aa, amino acid.

Figure 4

Moderate enhancement of the reconstructed heme biosynthesis pathway. a) Enhancement of key steps in heme biosynthesis (Hem2p, Hem3p, and Hem4p) using multi‐enzyme assembly. Hem2p, Hem3p, and Hem4p were fused with specific ligands and fixed to protein scaffolds driven by the constitutive promoters P _GAP (medium strength, HEME‐4 strain) and P_G7 (weak, HEME‐5 strain).^{[ 46 ]} The HEME‐3 strain without the synthetic protein scaffold was used as the control. b) The effects of overexpressing HEM13 (HEME‐7 strain) and deleting HMX1 (HEME‐8 strain) on the intracellular accumulation of heme. HEME‐6 indicates the control strain. c) Comparison between the final heme‐supplying strain HEME‐9 and the original strain X33‐Δku70 in heme biosynthesis. Data presented as mean values ± SD from three independent biological replicates (n = 3). Statistical evaluation (p‐value) compared to the control strain was conducted by a two‐tailed t‐test. *p < 0.05, **p < 0.01, ***p < 0.001 and NS representing non‐significance (p ≥ 0.05). Abbreviations: Scaf, scaffold; Lig, ligand; Dom, domain.

Figure 5

The biochemical properties of biosynthetic hemoproteins. a) The expression of several hemoproteins in the P1H9 strain. CK stands for the cell lysate of the X33‐Δku70 strain without the hemoprotein gene, and M stands for protein ladder. b) The spectral characteristics of purified hemoproteins. c) Detection of the heme‐binding ratio of purified hemoproteins using the difference spectrum between reduced and oxidized samples.^{[ 54 ]} d) The specific POD activity of purified P‐Mb and S‐Hb. e) The time‐course oxygen binding behavior of V‐Hb in the presence of sodium dithionite (reductant and oxygen scavenger). S _at.O2 stands for oxygen saturation degree.^{[ 55 ]} f) HPLC analysis of the catalytic efficiency of cells expressing BM3_mut in converting phenol to hydroquinone. Data presented as mean values ± SD from three independent biological replicates (n = 3). Statistical evaluation (p‐value) compared to the control strain was conducted by a two‐tailed t‐test. *p < 0.05, **p < 0.01, ***p < 0.001 and NS representing non‐significance (p ≥ 0.05).