Engineering the green algae Chlamydomonas incerta for recombinant protein production

Abstract

Chlamydomonas incerta, a genetically close relative of the model green alga Chlamydomonas reinhardtii, shows significant potential as a host for recombinant protein expression. Because of the close genetic relationship between C. incerta and C. reinhardtii, this species offers an additional reference point for advancing our understanding of photosynthetic organisms, and also provides a potential new candidate for biotechnological applications. This study investigates C. incerta's capacity to express three recombinant proteins: the fluorescent protein mCherry, the hemicellulose-degrading enzyme xylanase, and the plastic-degrading enzyme PHL7. We have also examined the capacity to target protein expression to various cellular compartments in this alga, including the cytosol, secretory pathway, cytoplasmic membrane, and cell wall. When compared directly with C. reinhardtii, C. incerta exhibited a distinct but notable capacity for recombinant protein production. Cellular transformation with a vector encoding mCherry revealed that C. incerta produced approximately 3.5 times higher fluorescence levels and a 3.7-fold increase in immunoblot intensity compared to C. reinhardtii. For xylanase expression and secretion, both C. incerta and C. reinhardtii showed similar secretion capacities and enzymatic activities, with comparable xylan degradation rates, highlighting the industrial applicability of xylanase expression in microalgae. Finally, C. incerta showed comparable PHL7 activity levels to C. reinhardtii, as demonstrated by the in vitro degradation of a polyester polyurethane suspension, Impranil® DLN. Finally, we also explored the potential of cellular fusion for the generation of genetic hybrids between C. incerta and C. reinhardtii as a means to enhance phenotypic diversity and augment genetic variation. We were able to generate genetic fusion that could exchange both the recombinant protein genes, as well as associated selectable marker genes into recombinant offspring. These findings emphasize C. incerta's potential as a robust platform for recombinant protein production, and as a powerful tool for gaining a better understanding of microalgal biology.

Fig 1. Cellular localization of mCherry to the cytosol, cell membrane, and cell wall.

Fluorescent microscopy demonstrated mCherry expression in the B) cytosol, C) secretory pathway, D) cell membrane, and E) cell wall. Scale bars indicate 5 μm. F, G, H) The recrystallized cell wall components of transgenic C. incerta pJPW2mCherry showed mCherry fluorescence, absent on the wild type. The recombinant strains were deposited at Chlamydomonas collection (Ci_pAH04mCherry CC-6206; Ci_pJP32mCherry CC-6207; Ci_pJPM1 CC-6208; Ci_pJPW2 CC-6209).

Fig 2. C. incerta exhibits higher secretion and expression of mCherry compared to C. reinhardtii. A) mCherry fluorescence readings at excitation wavelength of 580/9 nm and emission wavelength of 610/20 nm for C. incerta (n = 84) and C. reinhardtii (n = 84) demonstrated a 3.5-fold higher fluorescence intensity for C. incerta than C reinhardtii (p-value < 2.2 x 10^-16). Fluorescence readings were normalized with cell density using chlorophyll fluorescence. Threshold for positive expression (blue dashed line at 9.10 x 10^-3 RFU for C. incerta and green dashed line at 0.580 RFU for C. reinhardtii) was defined as three standard deviations above the mean activity level observed in the wild types. B) Immunoblot analysis using an anti-RFP antibody indicated the expected band size of mCherry at approximately 30 kDa for both C. incerta and C. reinhardtii, with a 3.76-fold higher band intensity for C. incerta. C) Fluorescent microscopy images showed mCherry signals inside vesicles localized within the cell’s secretory pathway. D, E) Growth curves of the top-expressing clone indicated that mCherry secretion from the transgenic C. incerta line did not affect cell growth compared to transgenic C. reinhardtii lines and their respective wild types.

Fig 3. Xylanase-expressing C. incerta and C. reinhardtii lines capable of fluorogenic substrate hydrolysis and commercial-grade xylan degradation.

A) Transgenic C. incerta (n = 84) and C. reinhardtii (n = 84) lines expressing xylanase demonstrated a statistically significant difference in activity by measuring the moles of product formed (µmol/s) over time (p-value 0.00116) via DiFMUX2 hydrolysis. Wild-type C. incerta (n = 3) and C. reinhardtii (n = 6) have no statistically significant difference (adjusted p-value 1.000) Fluorescence readings were measured at excitation wavelength of 355/9 nm and emission wavelength of 458/20 nm, and normalized with cell density using chlorophyll relative fluorescence unit (RFU). Threshold for positive expression (dashed line at 5.7710^-5 µmol/s) was defined as three standard deviations above the mean activity level observed in the wild types. B) Two of the three highest xylanase-expressing lines from C. incerta (F1 and G3) and C. reinhardtii (D4 and G4) demonstrated the capability to degrade industrial-grade xylan after 9 days. Samples F1 and G3 from C. incerta exhibited a xylan degradation rate of 215.85 µg⋅min⋅L^-1 and 305.06 µg⋅min^-1⋅L^-1, respectively. Samples D4 and G4 from C. reinhardtii exhibited a xylan degradation rate of 382.14 µg⋅min^-1⋅L^-1 and 626.02 µg⋅min^-1⋅L^-1, respectively.

Fig 4. Comparable PHL7 activity levels between C. incerta and C. reinhardtii based on Impranil® DLN degradation assays.

A) After 7 days, 52 halos (51/81, 62.963%) were observed for C. incerta (left) and 61 halos (61/84, 72.619%) for C. reinhardtii (right) on the TAP agar plates containing 0.5% (v/v) Impranil® DLN. For both replica plates, wells A1 to G12 represent potential transformants, wells H1 to H6 represent the respective wild types, and wells H7 to H12 represent blanks. B) The Impranil® DLN activity assay demonstrated a statistically significant difference in activity (i.e., change in OD at 350 nm over 14 days) between the transgenic C. incerta and C. reinhardtii lines (p-value 1.918 x 10–6). For C. incerta and C. reinhardtii, 23.457% (19/81) and 53.762% (46/84), respectively, of the colonies displayed a negative absorbance slope. Threshold for positive expression (blue dashed line at ∆OD -0.139 for C. incerta and green dashed line at ∆OD -0.113 for C. reinhardtii) was defined as three standard deviations below the mean activity level observed in the wild types.

Fig 5. Hybridizations of C. incerta holding bleomycin resistance and C. reinhardtii holding hygromycin B resistance.

A) Hybridizations were performed and 129 colonies were obtained on TAP agar plates containing 15 μg/mL zeocin and 30 μg/mL hygromycin. B) Four hybrids (D11, D9, E2, C10) that exhibited resistance to zeocin and hygromycin (and the parent strains) were plated onto TAP agar plates containing only 15 μg/mL zeocin, only 30 μg/mL hygromycin, or both 15 μg/mL zeocin and 30 μg/mL hygromycin. C) PCR analyses for the presence of bleomycin and hygromycin B resistance genes in the four hybrids, but amplicons for only 3 hybrids (D11, D9, C10) were obtained. The expected sizes of the PCR products were 500 bps for bleomycin and 162 bps for hygromycin B.