# Metabolic rewiring and biomass redistribution enable optimized mixotrophic growth in Chlamydomonas

## TL;DR

This study shows how Chlamydomonas algae optimize growth by reorganizing metabolism when using both light and acetate, reducing the cost of protein synthesis.

## Contribution

The paper reveals a novel metabolic synergy in mixotrophic Chlamydomonas growth through isotopic flux analysis, contradicting prior models.

## Key findings

- Acetate induces the glyoxylate cycle and suppresses gluconeogenesis in mixotrophic Chlamydomonas.
- Mixotrophic growth reduces photosynthetic flux and lowers protein synthesis costs for optimized growth.
- INST-MFA results show metabolic rewiring that enhances carbon conservation and biomass production.

## Abstract

Algae are a promising source of renewable feedstocks for energy, natural products, and biomass. Still, carbon partitioning strategies that dictate algae growth and biomass composition remain incompletely understood, limiting rational engineering efforts. Isotope-assisted metabolic flux analysis enables reconstruction of metabolic networks based on empirically deduced reaction rates. Using this approach, we compared photosynthetic and mixotrophic metabolism in the green alga Chlamydomonas and observed a metabolic synergy arising from acetate utilization in light. Acetate induced the glyoxylate cycle and, despite omics data and prior modeling predictions, suppressed gluconeogenesis while also reducing photosynthetic flux. Further, our flux maps illustrate how partial suppression of photosynthesis by acetate may help achieve growth optimization by reducing the costly burden of protein synthesis.

Aquatic photosynthetic systems account for approximately one-half of all global carbon assimilation and could be a significant source of renewable fuels and feedstocks. However, rapid growth and biomass production in algae have not always translated into high product yields, partly because central metabolism is context specific, with metabolic fluxes being influenced by nutrient conditions and other environmental factors. In the green microalga Chlamydomonas reinhardtii (Chlamydomonas), mixotrophic cultures (acetate + light) grow far faster than phototrophic (light only) or heterotrophic (acetate + dark) cultures, even though acetate partially suppresses photosynthesis. Here, an isotopic dilution strategy with unlabeled acetate was combined with 13CO2 transient labeling to perform isotopically nonstationary metabolic flux analysis (INST-MFA) and to directly compare autotrophic and mixotrophic metabolism in Chlamydomonas supported by data from transcriptomics, proteomics, and metabolomics. INST-MFA indicated that acetate induces a synergistic rewiring of metabolism, conserving carbon by using the glyoxylate cycle and suppressing gluconeogenesis, the latter of which was discordant with omics results and prior models. Additionally, our data provide a plausible rationale for the well-known suppression of photosynthesis by acetate. We propose that reduced total protein content in mixotrophic versus phototrophic cells, much of which is attributed to reduced levels of photosynthetic proteins, decreases the costly metabolic burden of protein synthesis and represents a growth rate optimization strategy.