Image_5_Exploiting photosynthesis-driven P450 activity to produce indican in tobacco chloroplasts.jpeg

Photosynthetic organelles offer attractive features for engineering small molecule bioproduction by their ability to convert solar energy into chemical energy required for metabolism. The possibility to couple biochemical production directly to photosynthetic assimilation as a source of energy and substrates has intrigued metabolic engineers. Specifically, the chemical diversity found in plants often relies on cytochrome P450-mediated hydroxylations that depend on reductant supply for catalysis and which often lead to metabolic bottlenecks for heterologous production of complex molecules. By directing P450 enzymes to plant chloroplasts one can elegantly deal with such redox prerequisites. In this study, we explore the capacity of the plant photosynthetic machinery to drive P450-dependent formation of the indigo precursor indoxyl-β-D-glucoside (indican) by targeting an engineered indican biosynthetic pathway to tobacco (Nicotiana benthamiana) chloroplasts. We show that both native and engineered variants belonging to the human CYP2 family are catalytically active in chloroplasts when driven by photosynthetic reducing power and optimize construct designs to improve productivity. However, while increasing supply of tryptophan leads to an increase in indole accumulation, it does not improve indican productivity, suggesting that P450 activity limits overall productivity. Co-expression of different redox partners also does not improve productivity, indicating that supply of reducing power is not a bottleneck. Finally, in vitro kinetic measurements showed that the different redox partners were efficiently reduced by photosystem I but plant ferredoxin provided the highest light-dependent P450 activity. This study demonstrates the inherent ability of photosynthesis to support P450-dependent metabolic pathways. Plants and photosynthetic microbes are therefore uniquely suited for engineering P450-dependent metabolic pathways regardless of enzyme origin. Our findings have implications for metabolic engineering in photosynthetic hosts for production of high-value chemicals or drug metabolites for pharmacological studies.</p

Table_1_Exploiting photosynthesis-driven P450 activity to produce indican in tobacco chloroplasts.docx

Photosynthetic organelles offer attractive features for engineering small molecule bioproduction by their ability to convert solar energy into chemical energy required for metabolism. The possibility to couple biochemical production directly to photosynthetic assimilation as a source of energy and substrates has intrigued metabolic engineers. Specifically, the chemical diversity found in plants often relies on cytochrome P450-mediated hydroxylations that depend on reductant supply for catalysis and which often lead to metabolic bottlenecks for heterologous production of complex molecules. By directing P450 enzymes to plant chloroplasts one can elegantly deal with such redox prerequisites. In this study, we explore the capacity of the plant photosynthetic machinery to drive P450-dependent formation of the indigo precursor indoxyl-β-D-glucoside (indican) by targeting an engineered indican biosynthetic pathway to tobacco (Nicotiana benthamiana) chloroplasts. We show that both native and engineered variants belonging to the human CYP2 family are catalytically active in chloroplasts when driven by photosynthetic reducing power and optimize construct designs to improve productivity. However, while increasing supply of tryptophan leads to an increase in indole accumulation, it does not improve indican productivity, suggesting that P450 activity limits overall productivity. Co-expression of different redox partners also does not improve productivity, indicating that supply of reducing power is not a bottleneck. Finally, in vitro kinetic measurements showed that the different redox partners were efficiently reduced by photosystem I but plant ferredoxin provided the highest light-dependent P450 activity. This study demonstrates the inherent ability of photosynthesis to support P450-dependent metabolic pathways. Plants and photosynthetic microbes are therefore uniquely suited for engineering P450-dependent metabolic pathways regardless of enzyme origin. Our findings have implications for metabolic engineering in photosynthetic hosts for production of high-value chemicals or drug metabolites for pharmacological studies.</p

Image_4_Exploiting photosynthesis-driven P450 activity to produce indican in tobacco chloroplasts.jpeg

Photosynthetic organelles offer attractive features for engineering small molecule bioproduction by their ability to convert solar energy into chemical energy required for metabolism. The possibility to couple biochemical production directly to photosynthetic assimilation as a source of energy and substrates has intrigued metabolic engineers. Specifically, the chemical diversity found in plants often relies on cytochrome P450-mediated hydroxylations that depend on reductant supply for catalysis and which often lead to metabolic bottlenecks for heterologous production of complex molecules. By directing P450 enzymes to plant chloroplasts one can elegantly deal with such redox prerequisites. In this study, we explore the capacity of the plant photosynthetic machinery to drive P450-dependent formation of the indigo precursor indoxyl-β-D-glucoside (indican) by targeting an engineered indican biosynthetic pathway to tobacco (Nicotiana benthamiana) chloroplasts. We show that both native and engineered variants belonging to the human CYP2 family are catalytically active in chloroplasts when driven by photosynthetic reducing power and optimize construct designs to improve productivity. However, while increasing supply of tryptophan leads to an increase in indole accumulation, it does not improve indican productivity, suggesting that P450 activity limits overall productivity. Co-expression of different redox partners also does not improve productivity, indicating that supply of reducing power is not a bottleneck. Finally, in vitro kinetic measurements showed that the different redox partners were efficiently reduced by photosystem I but plant ferredoxin provided the highest light-dependent P450 activity. This study demonstrates the inherent ability of photosynthesis to support P450-dependent metabolic pathways. Plants and photosynthetic microbes are therefore uniquely suited for engineering P450-dependent metabolic pathways regardless of enzyme origin. Our findings have implications for metabolic engineering in photosynthetic hosts for production of high-value chemicals or drug metabolites for pharmacological studies.</p

S2 Fig -

(A) Alphafold2 model of CnCel1, turned 180 on the y-axis compared to Fig 1B. CnCel1 is shown in magenta. CnCel1 aromatic residues potentially involved in substrate binding are shown as stick representation in blue and CnCel1 cysteines in yellow. The two unpaired cysteines are indicated. (B) Representative coomasie stained polyacrylamide gel of the expression supernatant, purified, and deglycosylated Cel1Cat protein. (C-D) Quantitative amino acids acid analysis of deglycosylated Cel1Cat confirms expression of correct protein. (E) Progress curves for oxidation of reduced fluorescein by non-enzyme bound copper and purified Cel1cat without added copper. Activity measured with relative fluorescent units (RFU) at 528 nm for 30 min. Free CuCl2 was tested as a negative control in stoichiometric amounts to a 5 μM copper loaded Cel1cat (3.75 μM). Cel1cat concentrations as indicated, without copper loading, was tested to assess initial copper loading from expression host. Reaction conditions are 75 mM phosphate citrate, pH 7.4, 25°C, Cel1cat blank (black line), 100 μM DHA (red line), 100 μM H2O2 (blue), or both (green line) was added to investigate LPMO copper reduction. Experiments done in triplicates, standard deviations shown, but not visible. (TIF)</p

<i>Cn</i>Cel1 affects melanization and capsule architecture.

(A) Melanization in the absence and presence of exogenous copper. Indicated strains were incubated on L-DOPA plates at 30°C or 37°C, with and without supplementation with exogenous CuSO4. Melanin formation was assessed at 1–2 days as indicated. (B-E) Surface capsule formation and architecture. The WT and cel1Δ mutant were incubated for 3 days in CIM tissue culture medium to induce capsule. Surface capsule characteristics were assessed by (B) India ink counter-staining and (C) SEM. Representative images are shown from 3 independent experiments. Quantification of capsule size (D) and cell body size (E) was performed using ImageJ/Fiji from static images of india ink-stained cells. Mean values from >100 cells per sample (+/- SEM) from 3 biological replicates are shown as bar graph. Data were plotted, and an unpaired t-test was performed using GraphPad Prism. (F) Electromobility of exopolysaccharide. The WT and cel1Δ mutant were incubated in CIM for 3 days. Exopolysaccharide in the culture supernatant was assessed for relative size and gel motility by agarose gel electrophoresis, transferred to a Nylon membrane, and probed with anti-GXM antibody 18B7. Representative image of multiple replicates is shown.</p

S1 Fig -

(A) Schematic domain overview of CnCel1 protein variants. The N-terminal signal peptide is highlighted in red, putative copper binding amino acids are highlighted in blue and labelled, and the unstructured C-terminus is highlighted in dark green. (B) Structure based alignment of Cel1 and the AA9 sequences from the phylogenetic analysis. The alignment was created using EXPRESSO of the T-COFFEE alignment package [85]. The final alignment was prepared using the ESPript 3.0 web server [90]. The substrate binding regions, L2, L3, and LC, are indicated, as well as the two paired cysteine disulfide bridges and two unpaired cysteines from the two protein fold models. (TIF)</p

<i>Cn</i> strains used in this study.

Fungi often adapt to environmental stress by altering their size, shape, or rate of cell division. These morphological changes require reorganization of the cell wall, a structural feature external to the cell membrane composed of highly interconnected polysaccharides and glycoproteins. Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that are typically secreted into the extracellular space to catalyze initial oxidative steps in the degradation of complex biopolymers such as chitin and cellulose. However, their roles in modifying endogenous microbial carbohydrates are poorly characterized. The CEL1 gene in the human fungal pathogen Cryptococcus neoformans (Cn) is predicted by sequence homology to encode an LPMO of the AA9 enzyme family. The CEL1 gene is induced by host physiological pH and temperature, and it is primarily localized to the fungal cell wall. Targeted mutation of the CEL1 gene revealed that it is required for the expression of stress response phenotypes, including thermotolerance, cell wall integrity, and efficient cell cycle progression. Accordingly, a cel1Δ deletion mutant was avirulent in two models of C. neoformans infection. Therefore, in contrast to LPMO activity in other microorganisms that primarily targets exogenous polysaccharides, these data suggest that CnCel1 promotes intrinsic fungal cell wall remodeling events required for efficient adaptation to the host environment.</div

<i>CEL1</i> is induced during alkaline pH and high temperature stress in a Rim101-dependent manner and is associated with the <i>Cn</i> cell wall.

(A) Cel1-4xFLAG protein expression assessed by western blot. The strain expressing the Cel1-4xFLAG fusion protein was incubated in SC medium (starting OD600 of 0.4) for 3h at indicated pH and temperature, or in SC medium supplemented with 1mM CuSO4 or 1mM BCS as indicated. Protein extraction was performed by TCA-based protein extraction. For each condition, 20 μL of crude TCA extract was analyzed by Western blot for Cel1-4xFLAG using the αFLAG-HRP conjugate antibody. The αH3 (anti-histone 3) antibody was used as a loading control. (B) Transcript abundance of Cn CEL1, CAP60, and two other predicted Cn LPMOs (CNAG_03405 and CNAG_07314). Previously reported RNASeq data was analyzed to quantify alterations in the expression of these four genes in response to changes in pH, as well as in two mutants impaired for growth at high pH (sre1Δ and rim101Δ mutant strains): Brown et al., 2020 [29] (WT pH8 vs. pH4 and pH8 sre1Δ vs. WT) and Brown et al., 2018 [28] (Tissue culture (TC) rim101Δ vs. WT). Transcript data were plotted with GraphPad Prism and are shown in log2-fold change. (C) Cel1-4xFLAG protein expression analysis by western blot in response to alkaline pH stress in the WT and rim101Δ strain. The two strains were incubated at 30°C in SC medium pH 8.15 (starting OD600 of 0.4) for indicated times, followed by TCA-based protein extraction. For each condition, 25 μL of crude TCA extract was analyzed by western blot for Cel1-4xFLAG expression using the αFLAG-HRP conjugate and αH3 as loading control. (D) Temperature-dependent Cel1-4xFLAG protein expression at alkaline pH. The strain expressing the Cel1-4xFLAG fusion protein was incubated in SC medium pH 8.15 (starting OD600 of 0.4) for 3h at indicated temperatures, followed by TCA-based protein extraction. For each condition, 25 μL of crude TCA extract was analyzed by western blot for Cel1-4xFLAG expression using the αFLAG-HRP conjugate and αH3 as loading control. (E) Cel1-4xFLAG protein expression in tissue culture medium. Two independent strains expressing the Cel1-4xFLAG fusion protein and the WT strain were incubated in either SC medium at 30°C or in CO2-independent tissue culture medium (CIM) at 37°C for 24 h (starting OD600 of 0.1), followed by TCA-based protein extraction. For each condition, 20 μL of crude TCA extract was analyzed by western blot for Cel1-4xFLAG expression using the αFLAG-HRP conjugate and αH3 as loading control. (F) Western blot analysis of Cel1-4xFLAG secretion pattern. Two independent strains expressing the Cel1-4xFLAG protein were incubated in CIM (starting OD600 of 0.1) for 24h. Cell pellets (P) and supernatant (SN) were harvested, and the supernatant was filtered (0.2μ filter) to remove residual cells. Total protein from each sample was extracted using the TCA-based protein extraction method. For each condition, 20 μL of crude TCA extract was analyzed by western blot for Cel1-4xFLAG using the αFLAG-HRP conjugate and αH3 as loading control. (G-H) Western blot analysis of Cel1-4xFLAG (G) and Sec63-GFP (H) protein levels upon cell wall degradation. Cel1-4xFLAG expression was induced as described in (F). Sec63-GFP was grown overnight in YPD at 30°C and then cultivated for 3h in SC+250μM CuSO4 at 30°C. Cell wall degradation was performed using increasing amounts of Zymolyase (10u, 50 u, 100u, 200u). As control for Cel1-4xFLAG and Sec63-GFP protein levels in the intact cell wall, the assay was performed without the addition of Zymolyase (0u sample). Total protein from each sample was extracted using the TCA-based protein extraction method. For each condition, 20 μL of crude TCA extract was analyzed by western blot for Cel1-4xFLAG using the αFLAG-HRP conjugate (G) and for Sec63-GFP (H) using the αGFP antibody. Histone3 levels were determined using the αH3 antibody.</p

<i>cel1Δ</i> shows cell cycle defects under host like stress conditions.

(A-D)In vitro titanization. The indicated strains were incubated overnight in YNB medium, diluted to OD600 of 0.001 in titan cell-inducing medium (PBS+10% HI-FBS, 37°C, 5%CO2) [49], and further incubated for 24h, 48h, and 72h. Cells were analyzed by microscopy and cell diameter measured using Image J/Fiji. (A-C) Scatter plots and bar charts of cell size distribution are demonstrated for indicated times. All graphs were generated using GraphPad Prism. (D) Representative DIC images of indicated strains after 72h growth in titan cell-inducing conditions. (E) Budding index (ratio of budded:total cells) of indicated strains after 18h of conditioning in YPD at 30°C or CIM at 37°C. A minimum of 300 cells were assessed per strain and condition. Shown is the mean +/- SEM of 5 biological replicates. Data were plotted using GraphPad Prism. A 2-way ANOVA was performed from log transformed data. (F) Average cell size of indicated strains after 18h of conditioning in YPD at 30°C or CIM at 37°C. A minimum of 150 cells was measured per strain and condition. Shown is the mean +/- SEM of 4 biological replicates. Data were plotted using GraphPad Prism. A 2-way ANOVA was performed from log transformed data. (G) Ploidy analysis in host-mimicking conditions. Indicated strains were inoculated to a starting OD600 of 0.1 and incubated for 24h in YPD at 30°C or CIM at 37°C. Cells were fixed and stained with propidium iodine. Propidium iodine staining was quantified by Flow Cytometry. Data were analyzed using the FlowJo software. A representative histogram is shown from 3 independent ploidy analyses.</p

Plasmids used in this study.

Fungi often adapt to environmental stress by altering their size, shape, or rate of cell division. These morphological changes require reorganization of the cell wall, a structural feature external to the cell membrane composed of highly interconnected polysaccharides and glycoproteins. Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that are typically secreted into the extracellular space to catalyze initial oxidative steps in the degradation of complex biopolymers such as chitin and cellulose. However, their roles in modifying endogenous microbial carbohydrates are poorly characterized. The CEL1 gene in the human fungal pathogen Cryptococcus neoformans (Cn) is predicted by sequence homology to encode an LPMO of the AA9 enzyme family. The CEL1 gene is induced by host physiological pH and temperature, and it is primarily localized to the fungal cell wall. Targeted mutation of the CEL1 gene revealed that it is required for the expression of stress response phenotypes, including thermotolerance, cell wall integrity, and efficient cell cycle progression. Accordingly, a cel1Δ deletion mutant was avirulent in two models of C. neoformans infection. Therefore, in contrast to LPMO activity in other microorganisms that primarily targets exogenous polysaccharides, these data suggest that CnCel1 promotes intrinsic fungal cell wall remodeling events required for efficient adaptation to the host environment.</div