Docosahexaenoic acid (DHA, 22:6ω-3), a principal ω-3 polyunsaturated fatty acid (PUFA) of human retina and brain membranes, plays essential roles in infant neurological and cognitive development and general human health [1,2]. DHA is traditionally extracted from deep-sea fish oil. However, development of sustainable alternative sources of DHA is highly desirable in view of instability of marine resources, and increasing incidence of environmental pollution [3]. Schizochytrium sp., a fast-growing heterotrophic marine thraustochytrid (single-celled saprotrophic eukaryote), has ⁓35% of total fatty acids (TFAs) consisting of DHA, and is a promising DHA production source [4].

Basic leucine zipper (bZIP) proteins, one of the largest families of transcription factors in eukaryotic cells, occur widely in animals, plants, and microorganisms [5,6]. They are characterized by a basic region followed by a leucine zipper coiled-coil region. The basic region is a highly conserved domain containing an N-X7-R/K-X9 motif, responsible for binding specific DNA. The leucine zipper region is a less conserved region containing a heptad repeating pattern of leucine or other hydrophobic amino acids (isoleucine, valine, phenylalanine, methionine) essential for dimerization [7,8]. bZIP transcription factors form homodimers or heterodimers with other bZIP transcription factors, which bind to short palindromic or pseudo-palindromic sequences of target genes [[9], [10], [11], [12]]. They are involved in regulation of a variety of physiological processes, including growth and aging, secondary metabolism, virulence and pathogenicity, and responses to abiotic and biotic stresses [[12], [13], [14], [15], [16], [17]]. bZIP transcription factor AflRsmA, found in the saprotrophic, pathogenic fungus Aspergillus flavus, mediates oxidative stress response, virulence, sclerotia production, and aflatoxin biosynthesis [14]. In Penicillium chrysogenum, bZIP transcription factor PcYap1 senses the presence of H2O2-generated reactive oxygen species (ROS), undergoes conformational change by forming intramolecular disulfide bonds, and becomes localized in the nucleus. Oxidized PcYap1 binds to 5′-TTA(G/C)TAA-3′ motif of target genes, and then activates conidiation (asexual reproduction of filamentous fungi from spores), penicillin biosynthesis, and ROS responses [12]. Another Yap-like bZIP protein in P. chrysogenum, PcRsmA, also helps mediate these processes [12]. There have been few functional studies of bZIP transcriptional regulators in microorganisms.

In Schizochytrium, the major components of TFAs are DHA, hexadecanoic acid (C16:0), tetradecanoic acid (C14:0), and docosapentaenoic acid (DPA; C22:5ω-6). A single large fatty acid synthase (FAS; encoded by fas gene) is responsible for synthesis of saturated fatty acids (SFAs) C14:0 and C16:0, and a polyketide synthase-like PUFA synthase is responsible for de novo synthesis of DHA and DPA [18,19]. The PUFA synthase complex is encoded by three open reading frames: pfa1, pfa2, and pfa3 [[19], [20], [21]]. Studies to date of Schizochytrium have focused mainly on enhancement of DHA production through traditional methods [[22], [23], [24], [25]] or genetic engineering [[26], [27], [28]]. Our knowledge of regulation of DHA biosynthesis and lipid accumulation in this genus is limited and fragmentary; this presents an obstacle to its development as an industrial platform for production of PUFAs or oleochemicals.

Lipid accumulation in oleaginous microorganisms is regulated by complex regulatory networks [29,30]. Lipid production can be increased by engineering of regulatory genes that govern lipid accumulation. In the ascomycetous yeast Yarrowia lipolytica, Ylsnf1 deletion resulted in a 2.6-fold increase of fatty acid titer and 52% increase in eicosapentaenoic acid (EPA) titer [31]. In the microalgae Nannochloropsis gaditana, lipid production was increased 2-fold by deletion of a single Zn(II)2Cys6-encoding gene [32]. Elucidation of the transcriptional regulatory network governing lipid accumulation in Schizochytrium will presumably provide a basis for rational construction of DHA high-yielding strains.

Schizochytrium are heterotrophic marine microorganisms having major functional roles as decomposers and PUFA producers in coastal marine ecosystems [33]. Our 2022 study revealed that C2H2 zinc finger transcription factor LipR is a key repressor of DHA and lipid production in Schizochytrium [34]; aside from this, essentially nothing is known regarding regulators that control DHA biosynthesis or stress responses in this genus or other thraustochytrids. We describe here identification in Schizochytrium of a novel bZIP transcription factor, termed FabR (fatty acid biosynthesis repressor), that represses DHA biosynthesis and lipid accumulation by directly regulating transcription of PUFA synthase and FAS genes, and also mediates H2O2 stress response and represses transcription of H2O2-scavenging genes, fas and pfa genes in a redox-dependent manner. Deletion of fabR (generating strain ΔfabR) promoted cell growth, enhanced tolerance to H2O2 stress, and increased DHA yield 46.5%. ΔfabR has strong potential for improved DHA production.