Enhancing carbohydrate repartitioning into lipid and carotenoid by disruption of microalgae starch debranching enzyme

Abstract

Light/dark cycling is an inherent condition of outdoor microalgae cultivation, but is often unfavorable for lipid accumulation. This study aims to identify promising targets for metabolic engineering of improved lipid accumulation under outdoor conditions. Consequently, the lipid-rich mutant Chlamydomonas sp. KOR1 was developed through light/dark-conditioned screening. During dark periods with depressed CO₂ fixation, KOR1 shows rapid carbohydrate degradation together with increased lipid and carotenoid contents. KOR1 was subsequently characterized with extensive mutation of the ISA1 gene encoding a starch debranching enzyme (DBE). Dynamic time-course profiling and metabolomics reveal dramatic changes in KOR1 metabolism throughout light/dark cycles. During light periods, increased flux from CO₂ through glycolytic intermediates is directly observed to accompany enhanced formation of small starch-like particles, which are then efficiently repartitioned in the next dark cycle. This study demonstrates that disruption of DBE can improve biofuel production under light/dark conditions, through accelerated carbohydrate repartitioning into lipid and carotenoid.

Fig. 1. Time-course profiles of nitrogen consumption and energy storage.

a Residual nitrate concentration in the culture media. b Biomass. c Lipid content. d Carbohydrate content. White and gray bands represent light and dark periods, respectively. Error bars indicate the standard deviation of three replicate experiments.

Fig. 2. Mutations in the ISA1 gene of Chlamydomonas sp. KOR1.

Chlamydomonas sp. ISA1 encodes an isoamylase-type starch debranching enzyme (DBE), with an N-terminal early set domain and C-terminal α-amylase catalytic domain. Predicted exons are shown as white rectangles. Deletion and insertion mutation sites in KOR1 ISA1 are shown as red bars.

Fig. 3. Cell morphology viewed by transmission electron microscopy (TEM).

Cells were harvested under nitrate-replete conditions (Day 1.5) and nitrate-deplete conditions (Day 10.0 and Day 10.5), fixed immediately, and visualized. Day 1.5 and Day 10.5 are just before the end of a light period (dusk) and Day 10.0 is just before the end of a dark period (dawn). S: starch granule, L: lipid droplet, P: pyrenoid, N: nucleus, PS: periplasmic space.

Fig. 4. Time-course profiles of photosynthetic pigments.

a Lutein content. b β-Carotene content. c Chlorophyll a+b content. White and gray bands represent light and dark periods, respectively. Error bars indicate the standard deviation of three replicate experiments.

Fig. 5. Diurnal profiles of the metabolic pool size.

ZT zeitgeber time, S7P sedoheptulose 7‐phosphate, Ru5P ribulose 5‐phosphate, RuBP ribulose-1,5-bisphosphate, 3PG 3‐phosphoglycerate, E4P erythrose 4-phosphate, F6P fructose 6‐phosphate, G6P glucose 6‐phosphate, G3P glycerol 3‐phosphate, PEP phosphoenolpyruvate, DXP 1-deoxy-d-xylulose 5-phosphate, MEcPP 2-C-methyl-d-erythritol-2,4-cyclopyrophosphate. Reactions containing single and multiple enzymatic steps are represented by solid and dotted lines, respectively. White and gray bands represent light and dark periods, respectively. Error bars indicate the standard deviation of three replicate experiments (*p < 0.05 by Welch’s t test).

Fig. 6. Dynamic metabolic profiles under illuminated conditions.

Cells grown under the light/dark conditions for 5.5 days were harvested and resuspended in medium containing NaH¹³CO₃. Vertical axes: level of ¹³C-labeled metabolites calculated by multiplying metabolic pool size with ¹³C-labeled ratio. Horizontal axes: labeling time after starting ¹³C supply to the cells. Error bars indicate the standard deviation of three replicate experiments (*p < 0.05 by Welch’s t test).

Fig. 7. Carbon partition/repartition model for DBE-deficient microalgae under light/dark conditions.

DBE-deficient microalgae accumulate carbohydrate as phytoglycogen, instead of starch. Under light conditions, carbon resources derived from CO₂ are partitioned into phytoglycogen. Under dark conditions, phytoglycogen is rapidly converted into intermediate metabolites and then repartitioned into lipid/carotenoid.