Engineered production of isoprene from the model green microalgaChlamydomonas reinhardtii

Abstract

Isoprene is a clear, colorless, volatile 5-carbon hydrocarbon that is one monomer of all cellular isoprenoids and a platform chemical with multiple applications in industry. Many plants have evolved isoprene synthases (IspSs) with the capacity to liberate isoprene from dimethylallyl pyrophosphate (DMAPP) as part of cellular protection mechanisms. Isoprene is hydrophobic and volatile, rapidly leaves plant tissues and is one of the main carbon emission sources from vegetation globally. The universality of isoprenoid metabolism allows volatile isoprene production from microbes expressing heterologous IspSs. Here, we compared heterologous overexpression from the nuclear genome and localization into the plastid of four plant terpene synthases (TPs) in the green microalga Chlamydomonas reinhardtii. Using sealed vial mixotrophic cultivation, direct quantification of isoprene production was achieved from the headspace of living cultures, with the highest isoprene production observed in algae expressing the Ipomoea batatas IspS. Perturbations of the downstream carotenoid pathway through keto carotenoid biosynthesis enhanced isoprene titers, which could be further enhanced by increasing flux towards DMAPP through heterologous co-expression of a yeast isopentenyl-PP delta isomerase. Multiplexed controlled-environment testing revealed that cultivation temperature, rather than illumination intensity, was the main factor affecting isoprene yield from the engineered alga. This is the first report of heterologous isoprene production from a eukaryotic alga and sets a foundation for further exploration of carbon conversion to this commodity chemical.

Bicistronic expression of nuclear transgenes in Chlamydomonas reinhardtii

“…An increase in overall expression was also observed on the C. reinhardtii β‐carotene ketolase ( Cr BKT)_YFP fusion expressed from a CS2_ aphVIII ‐containing construct (Figure S9). This protein is known to function at low levels as it has strong catalytic activity (Amendola et al., 2023; Cazzaniga et al., 2022; 2024; Perozeni et al., 2020; 2023), and here, its increase over MC control was noticeable across a population but not drastically improved (Figure S9).…”

A synthetic biology and green bioprocess approach to recreate agarwood sesquiterpenoid mixtures

“…32 That algae rely on this pathway for essential photosynthetic carotenoids implies an inherent capacity to channel carbon fixed by photosynthesis to isoprenoids, which could be tapped for heterologous terpene biosynthesis. The first reports of micrograms of heterologous terpenes per litre of algal culture 27 have now been superseded by hundreds of milligrams per litre, 25,33 owing to improved transgene design, 34,35 enhanced metabolic channelling, and high-cell-density cultivation strategies. 26,36 Here, we demonstrate for the first time a combinatorial modification of two metabolic pathways simultaneously in C. reinhardtii by perturbing plastid carotenoid and cytoplasmic sterol biosynthesis in the same strain (Fig.…”

“…which increased farnesyl pyrophosphate pools in the cytoplasm and led to improvements in heterologous bisabolene or patchoulol yields when their synthases were expressed. 24,26 Disruption of carotenoid homeostasis by introduction of a beta-carotene ketolase to biosynthesise ketocarotenoids in the plastid led to pronounced increases of heterologous isoprene biosynthesis 25 (Fig. 2a, right panel).…”

“…22,23 Enhanced heterologous terpenoid yields have been demonstrated from this green alga following metabolic engineering. [24][25][26] These include the knockdown of native squalene synthase (CrSQS-k.d.) which increased farnesyl pyrophosphate pools in the cytoplasm and led to improvements in heterologous bisabolene or patchoulol yields when their synthases were expressed.…”

A green alternative to fragrant agarwood sesquiterpenoid production

“…Chlamydomonas reinhardtii is an emerging host for metabolic engineering and heterologous terpene production 22,23 , as is known to have enhanced heterologous terpenoid yields following metabolic alterations [24][25][26] . These included the knockdown of native squalene synthase (CrSQS-k.d.)…”