High-level expression of leghemoglobin in Kluyveromyces marxianus by remodeling the heme metabolism pathway

Abstract

Soy leghemoglobin, when bound to heme, imparts a meat-like color and flavor and can serve as a substitute for animal-derived proteins. Enhancing cellular heme synthesis improves the recombinant expression of leghemoglobin in yeast. To achieve high-level expression of leghemoglobin A (LBA) in Kluyveromyces marxianus, a food-safe yeast, large-scale heme synthesis modules were transferred into K. marxianus using yeast artificial chromosomes (KmYACs). These modules contained up to 8 native and heterologous genes to promote the supply of heme precursors and downstream synthesis. Next, eight genes inhibiting heme or LBA synthesis were individually or combinatorially deleted, with the lsc1Δssn3Δ mutant yielding the best results. Subsequently, heme synthesis modules were combined with the lsc1Δssn3Δ mutant. In the resulting strains, the module genes were all actively expressed. Among these module genes, heterologous S. cerevisiae genes in the downstream heme synthesis pathway significantly enhanced the expression of their counterparts in K. marxianus, resulting in high heme content and LBA yield. After optimizing the medium recipe by adjusting the concentrations of glucose, glycine, and FeSO₄·7H₂O, a heme content of 66.32 mg/L and an intracellular LBA titer of 7.27 g/L were achieved in the engineered strain in a 5 L fermentor. This represents the highest intracellular expression of leghemoglobin in microorganisms to date. The leghemoglobin produced by K. marxianus can be utilized as a safe ingredient for plant-based protein products.

Keywords: Kluyveromyces marxianus; heme biosynthesis; high-level expression; leghemoglobin; yeast artificial chromosomes.

FIGURE 1

Correlation between heme synthesis and LBA production in K. marxianus. (A) Relative mRNA levels of the heme synthesis genes pathway in T1a and T1a-LBA. Cells were collected after culturing in YD medium for 48 h. The mRNA level of the heme synthesis gene was calculated relative to that of SWC4. Values represent the mean ± S.D. (n = 3). (B) Expression of LBA in T1a. pLBA was transformed into T1a, resulting in T1a-LBA. Cells were collected after culturing in YD medium for 72 h. The cell lysate was subjected to SDS-PAGE. Four independent clones (1–4) of T1a-LBA were analyzed. (C) Western of cell lysate of T1a and T1a-LBA-His₆. pLBA-His₆ was transformed into T1a, resulting in T1a-LBA-His₆. Cells were collected after culturing in YD medium for 72 h. The cell lysate was subjected to SDS-PAGE and Western blot. (D) Relative Content of heme and free porphyrin in T1a and T1a-LBA. Cells were cultured in YD for 72 h. The content in T1a was designated as 1. Values represent the mean ± S.D. (n = 3). ***, p < 0.001.

FIGURE 2

Remodeling of K. marxianus heme synthesis pathway by YAC. (A) Genes included in the heme synthesis module. (B) Flow chart of constructing KM-YAC that carried designed heme synthesis modules. KmYAC [C4+Down] and KmYAC [C4+C5+Down] were transformed into KM_DHW to obtain KM_C4D and KM_C45D, respectively. (C) Relative content of heme and free porphyrin in KM_ΔHW, KM_C4D, and KM_C45D. Cells were cultured in YD medium for 72 h. The content in KM_ΔHW was designated as 1. Values represent the mean ± S.D. (n = 3). **, p < 0.01. ns, not significant. (D) Relative levels of LBA in KM_ΔHW-LBA, KM_C4D-LBA and KM_C45D-LBA. pLBA was transformed into KM_ΔHW, KM_C4D, and KM_C45D, resulting in KM_ΔHW-LBA, KM_C4D-LBA and KM_C45D-LBA, respectively. The transformants were then cultured in YD medium for 72 h. The level of LBA in KM_ΔHW-LBA was designated as 1. Values represent the mean ± S.D. (n = 3). *, p < 0.05.

FIGURE 3

Improving heme synthesis and LBA production by deleting inhibitory genes. (A) Schematic diagram of effects of deletions. Deletions were expected to abolish the synthesis of succinate (lsc1Δ, lsc2Δ), repression of HEM13 (rox1Δ, hap1Δ, tup1Δ, ssn3Δ), or degradation of misfolded proteins (vps10Δ, pep4Δ). (B,C) Effect of deleting a single gene (B) or multiple genes (C) on the biomass, heme and free porphyrin content, and LBA production. Predicted effects of deletion(s) on enhancing 5-ALA synthesis, reducing repression of HEM13, and reducing protein degradation were checked. Genes were deleted in T1a. OD₆₀₀ and the total content of heme and free porphyrin in T1a and deletion mutant were compared after culturing in YD medium for 72 h. To compare the production of LBA, pLBA was transformed into T1a and mutants, respectively. The resulting transformants were cultured in YD medium for 72 h. Heme content and level of LBA in T1a were designated as 1 when compared to mutants. Values represent the mean ± S.D. (n = 3). *, p < 0.05, **, p < 0.01, ***, p < 0.001.

FIGURE 4

Expressions of heme synthesis genes in strains containing designed heme synthesis modules and deletions of inhibitory genes. (A–C) The relative mRNA levels of the heme synthesis genes pathway in KM_LSHW (A), KM_LSC4D (B), and KMLS_C45D (C). KmYAC [C4+Down] and KmYAC [C4+C5+Down] were transformed into KM_LSHW to obtain KM_LSC4D and KM_LSC45D, respectively. Cells were collected after culturing in YD medium for 72 h. The mRNA levels of heme synthesis genes were calculated relative to that of SWC4. The relative mRNA levels of genomic K. marxianus genes were represented by black columns, while those of the heterologous genes introduced by KmYAC were represented by white columns. The relative mRNA levels of K. marxianus genes expressed from one genomic copy and one additional copy introduced by KmYAC were represented by grey columns. The values represent the mean ± S.D. (n = 3).

FIGURE 5

The heme content and LBA production in strains containing designed heme synthesis modules and deletion of inhibitory genes. (A) The relative contents of heme and free porphyrin in KM_LSC4D and KM_LSC45D. Cells were cultured in YD medium for 72 h. The heme content in KM_LSHW was designated as 1. The values represent the mean ± S.D. (n = 3). **, p < 0.01, ***, p < 0.001. (B) The relative levels of LBA produced in KM_LSC4D and KM_LSC45D. pLBA was transformed into KM_LSHW, KM_LSC4D and KM_LSC45D, resulting in KM_LSHW-LBA, KM_LSC4D-LBA and KM_LSC45D-LBA, respectively. The transformants were then cultured in YD medium for 72 h. The level of LBA produced in KM_LSHW-LBA was designated as 1.

FIGURE 6

Stability of KmYAC and LBA-expressing plasmid in KM_LSC4D. KM_LSC4D-LBA were grown for 70 generations without selective pressure. The percentage of cells containing KmYAC [C4+Down] and pLBA was calculated after growing for 7, 14, 21, 42, 56, and 70 generations. The values represent the mean ± S.D. (n = 3). *, p < 0.05. **, p < 0.01. ns, not significant.

FIGURE 7

Optimization of the medium recipe and feed-batch fermentation for improved yield of LBA. (A) Optimization of the medium recipe to improve the yield of LBA. KM_LSC4D-LBA was grown in a medium with the indicated concentrations of glucose, glycine, and FeSO₄·7H₂O (Fe²⁺) for 72 h. The cell lysate was subjected to SDS-PAGE. The bands of LBA were scanned in grayscale, and the intensities of the bands were quantified. The values represent the mean ± S.D. (n = 3). (B) Growth curves of KM_LSC4D-LBA and T1a-LBA in a 5L fermentor. pLBA was transformed into T1a, resulting in T1a-LBA, which served as a control. Cells were grown in the optimized medium in a feed-batch 5L fermentor for 72 h. (C,D) Comparison of heme concentrations in KM_LSC4D-LBA and T1a-LBA during feed-batch fermentation. Cells were collected at the indicated times during feed-batch fermentation described in (B). The concentrations of heme in the cell lysate at each time point were quantified by HPLC and compared (C). A chromatogram map of 36-h samples was shown in (D). (E) Image of cells in the fermentor after 36 h. (F) SDS-PAGE of cell lysate of KM_LSC4D-LBA and T1a-LBA during feed-batch fermentation. Cells were collected at the indicated times during feed-batch fermentation described in (B). The cell lysate was diluted and subjected to SDS-PAGE. The amount of LBA was semi-quantified using a β-lactoglobulin standard. (G) Comparison of LBA titer in KM_LSC4D-LBA and T1a-LBA during feed-batch fermentation. The values represent the mean ± S.D. (n = 3). ***, p < 0.001.