Inducible two-step cultivation of microalgae: engineering the growth and lipid biosynthesis for biofuel production

1 Introduction

Over the past few decades, global energy demand has increased substantially to keep pace with the remarkable rate of technological advancement. The twenty-first century faces several critical global challenges, including food scarcity, overpopulation, climate change, and the progressive depletion of fossil fuel resources. At present, the world’s energy needs are primarily met by fossil fuels, which remain the dominant energy source worldwide (Maamoun et al., 2020). The most recent data shows that, excluding land use and forestry, worldwide GHG emissions in 2024 were 53.2 Gt CO₂eq. Compared to 2023 levels, the 2024 data showed a 1.3% increase, or 665 Mt CO₂eq (Crippa et al., 2025).

Among renewable energy sources, biofuels have received considerable attention as sustainable energy carriers and are broadly categorized into four generations based on feedstock types and technological advancements. Among them, third-generation biofuels are derived from microalgae and other microorganisms, which attracted significant attention for their high productivity, rapid growth rates, and minimal competition for arable land. The fatty acids produced by microalgae can be used to produce biodiesel, providing a renewable, biodegradable, non-toxic, and safe fuel. A recent report says that using biodiesel reduces carbon dioxide emissions by 78%, sulfur emissions by 98%, and particulate matter by 50% (Banerjee et al., 2016; Kumawat et al., 2024). Meanwhile, the fourth-generation biofuels are produced by integrating genetic and metabolic engineering strategies to improve the desired traits of biofuel-producing organisms. These approaches mainly focus on improving key traits of biofuel-producing organisms, including the ability to utilize diverse carbon sources (e.g., pentoses and hexoses), enhance lipid synthesis, and improve photosynthesis and carbon fixation. Currently, two approaches have been used: (i) engineering pathways in native producers to optimize growth rates, redirect metabolic flux toward biofuel production, and increase production yield; and (ii) reconstructing biosynthetic pathways identified in natural producers in more genetically accessible model organisms (Cavelius et al., 2023). Even though many oleaginous green algae, like chlorella and Scenedesmus, have much higher lipid content than traditional lipid-producing crops like maize, soybean, canola, castor, sunflower, and palm oil (Udayan et al., 2022). This high lipid content may actually lead to lower biomass productivity, and high biomass does not always imply high lipid productivity (Adams et al., 2013). As a result, focusing solely on lipid content rather than biomass is biased, as an inverse relationship exists between biomass productivity and lipid accumulation. Conventionally, nutrient limitation and environmental stress are commonly used techniques to increase lipid productivity, but they also affect growth, thereby reducing total biomass yield and productivity. Therefore, selecting resilient and adaptable microalgae strains is the first step towards large-scale stress-induced production, as strain selection, precise control of growth conditions, and PBR parameters all play important roles in optimizing biomass and lipid yields (Chu, 2017a).

In this context, two-step cultivation strategies have gained considerable attention, wherein microalgae are initially grown under optimal conditions to produce biomass, followed by the application of stress conditions to induce carbohydrate and lipid accumulation. This approach decouples the growth and lipid production phases, thereby significantly enhancing biofuel productivity and process efficiency (Piyatilleke et al., 2025). However, the economic viability of the two-step cultivation system still remains a concern. But this limitation can be mitigated by improving lipid-induction strategies and eliminating preharvesting steps. A wide range of physicochemical and nutritional triggers has been used to increase lipid accumulation and biomass productivity in microalgae (Chu, 2017b; Yang et al., 2018).

Though optimizing the timing of the second stage across strains for stress induction will provide targeted metabolite production, it will be time-consuming and requires prior knowledge of the microalgae. These limitations can be overcome by a well-defined, controllable, and clear induction system in the second stage, thereby enabling precise regulation and improved production of targeted metabolites. The following section discusses the inducible promoter system for microalgae, which can be applied in the two-step cultivation setup, with a major focus on available inducible systems and the molecular basis of promoter function for transformation and induction.

2 Two-step microalgae cultivation: key limitations

The two-step cultivation strategy has an initial phase in which cells are grown under ideal or nutrient-rich conditions, followed by a second phase in which specific stress conditions are imposed to drive the metabolic flux towards the synthesis of the desired molecule. This approach focused on achieving high biomass production in stage I, while stage II induced stress-mediated metabolic reprogramming to enhance the synthesis of desired metabolites, such as lipids, carbohydrates, and proteins (Nagappan et al., 2019). Several studies using the two-step cultivation system have highlighted its potential and limitations. For example, Perečinec et al (2022) demonstrated that precise selenite dosing and timing are important to prevent excessive ROS accumulation and cell death, unlike the tolerant lag-phase adaptation observed in a one-stage system (Perečinec et al., 2022). With the same approach, a two-step regulatory strategy was employed to enhance both lipid and biomass productivity, where supplementation with additional phosphorus and nitrogen by the end of the second day significantly improved lipid yield (Li et al., 2023). Despite these promising outcomes, the effectiveness of the two-step system depends primarily on prior knowledge of the strain’s physiology, growth conditions, and appropriate stress inducers.

Although extensive research has been conducted using the two-stage approach, obtaining consistent results is challenging and may fall short of the precision and efficiency achievable with genetically modified strains. Therefore, a deeper understanding of stress responses and metabolite induction is necessary to address these limitations. Further, several operational and physiological constraints also complicate the two-step cultivation system. A fundamental challenge lies in the trade-off between biomass accumulation and lipid production, where excessive or poorly timed stress treatments can suppress growth and reduce productivity (Shokravi et al., 2020). For example, high inoculum density in stage II can affects light penetration, thereby limiting photosynthetic efficiency and reducing metabolic flux towards the production of desired metabolites. While interventions such as artificial lighting or reduced culture depth can partially mitigate these effects, they will reflect an increase in production costs (Shokravi et al., 2020). For example, in a study performed by Singh and Sharma (2012), cultures started at 0.5 OD (680 nm) showed reduced light penetration and shading effects, whereas those started at 0.1 OD (680 nm) showed limited productivity due to insufficient initial biomass (Singh and Sharma, 2012). Other limitations, such as nitrogen limitation, restrict normal cellular metabolism and indirectly affect lipid productivity, even though it enhances lipid storage (Alishah Aratboni et al., 2019). Similarly, a study by Narala et al. (2016) showed that the assessed hybrid two-step cultivation system allows biomass production and in order to generate value-added products, one or more effective stress induction is required Further, the economic viability of the two-step cultivation system (TSCS) could be greatly increased by improving lipid-induction techniques and eliminating the pre-harvesting stage (Aziz et al., 2020; Narala et al., 2016) Additionally, stress treatments often favor the accumulation of neutral storage lipids at the expense of Polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA), thereby lowering the biodiesel quality. Further, scalability remains a critical concern, as most studies are conducted in laboratory levels, whereas large-scale production, particularly in open-pond systems, is associated with a high risk of contamination (Klein et al., 2024). Furthermore, not all algal species can effectively adapt to abrupt environmental shifts imposed during the phase transition. For example, the high salinity of the marine RAS wastewater used in Phase II severely harmed Chlorella sp. (Csp), leading to decreased biomass production and an inability to regain its maximal growth rate compared to marine-adapted strains (Villanova et al., 2023). And other concerns on measuring overall process performance must be evaluated in terms of total lipid productivity. Because in many cases, an increase in lipid percentage is counteracted by a stagnation or decrease in total biomass when the induction phase is excessively intense or prolonged, resulting in reduced overall yields, but the lipid percentage remains high.

To overcome these limitations on attaining high biomass density and lipid productivity, precise, tunable stress-induction strategies are essential in stage II. In this context, an inducible genetic switch system offers a promising alternative, enabling controlled activation of metabolic pathways in response to defined signals and limiting the off-target protein synthesis. The following section outlines the principles underlying such inducible systems and their mechanism of activation.

3 Inducible genetic switches: targeted solutions for phase II limitations

Synthetic biology aims to design programmable genetic circuits and regulatory networks within living systems to allow precise control over the biological host (Wang et al., 2013). These engineered frameworks allow cells to sense, integrate, and adapt to diverse internal and external stimuli. By modulating gene expression, cells can be programmed to achieve desired biological activities, such as increasing metabolites, biotherapeutic compounds, and reprogramming cellular behavior (Karataş and Furkan, 2025). The key regulatory elements used for this purpose are natural and synthetic promoters, which allow precise temporal and quantitative control of transcriptional activity. Figure 1 illustrates endogenous and exogenous promoters: native promoters are activated under environmental stress, whereas exogenous or synthetic promoters are expressed via engineered regulatory elements and an external inducer.

3.1 Natural inducible promoters

Natural promoters are endogenous cis-regulatory DNA sequences in an organism’s genome that regulate gene transcription and are classified into constitutive and inducible types. Constitutive promoters are essential for maintaining basic cellular processes and are expressed regardless of developmental stage, cell cycle state, or environmental conditions. For example, the photosystem I complex protein (psaD) and Rubisco small subunit 2 (RbcS2) are constitutive promoters (Kim et al., 2023). Meanwhile, inducible promoters are genetic switches that can be activated by environmental inducers, such as stress factors. In microalgae, both are important because constitutive promoters are useful for steady expression. Meanwhile, synthetic promoters are extended by assembling or modifying cis-regulatory elements to make expression tunable, predictable, or stronger than constitutive promoters. While constitutive promoters are commonly used for heterologous metabolite production, their uncontrolled nature can be harmful to host cells, thereby limiting the expression of foreign proteins and imposing a high metabolic burden. Further, the persistent overexpression of recombinant pathways can disrupt the cellular homeostasis, reduce growth, viability, and overall bioprocess efficiency (Ji et al., 2025). To address these limitations, dynamic regulation strategies offer a powerful alternative by allowing temporal decoupling of growth and production phases. Theoretical and experimental studies have demonstrated that growth and production can be separated by enabling cells to proliferate and then triggering a transition to chemical synthesis after a sizable population is achieved (Hoang et al., 2022). An effective implementation of dynamic control involves the use of substrate-dependent promoters or inducible promoters, which can be activated once a specific cell density is reached (Bateman et al., 2001). These promoters remain transcriptionally silent under basal conditions and are “triggered” or activated only upon exposure to defined signals such as nutrient limitation, osmotic stress, or chemical starvation, etc. By controlling the inducer availability in a culture environment, gene expression can be regulated, thereby minimize the cytotoxic effects associated with recombinant protein production, leading to improve productivity (He et al., 2023).

An inducible promoter system, such as the Lac operon, is a natural system that enables precise control of gene expression under specific environmental conditions, making it biotechnologically important. Their tightly and tunable expression is particularly advantageous when the expressed protein is cytotoxic to the cell in higher levels or imposes a significant metabolic burden on host cells. Based on the nature of activating stimulus, inducible promoters are broadly categorized into two types, chemically regulated or physiologically regulated systems. The chemically inducible promoters are activated or inhibited by the presence or absence of chemicals such as alcohols, antibiotics, hormones, or carbon sources. In contrast, physiologically regulated promoters respond to abiotic factors, such as osmotic stress, temperature, light, and other environmental factors (Kluge et al., 2018).

A well-known example of the chemical inducers, such as IPTG (Isopropyl-D-1-thiogalactopyranoside), is considered the most efficient means of inducing gene expression due to its tight regulation and rapid responsiveness. At the mechanistic level, inducible expression systems operate through transcriptional regulation mediated by repressor proteins that respond to specific ligands. For example, anhydrotetracycline (ATc) and isopropyl-β-d-thiogalactopyranoside (IPTG) can relieve the repression mediated by TetR and LacI, the two most commonly used repressor proteins (Zhang et al., 2022). In the tetracycline system, TetR binds to the tetO operator and inhibits expression of the tetracycline resistance gene in the absence of tetracycline. Meanwhile, when tetracycline is present, the binding affinity is greatly reduced, allowing expression of the resistance gene. However, their applications at an industrial scale are often limited due to high cost, potential toxicity, and challenges associated with their delivery and removal (Sadravi et al., 2026). To harness these inducer systems, using low-cost carbon sources or substrates as inducers may be a viable strategy for large-scale production. In parallel, the non-chemical induction systems, particularly those based on physical stimuli, are gaining increased attention. For example, in vitro studies have demonstrated that PIF3, a basic helix-loop-helix protein, specifically interacts with the photoactivated (Pfr) forms of phytochromes phyA and phyB upon light activation. This light-dependent, conformer-specific interaction has been successfully harnessed to create a light-inducible gene expression system (Shimizu-Sato et al., 2002).

The development of inducible promoters begins with the identification and isolation of a naturally responsive cis-regulatory sequence from native genome. For example, the cold-inducible, CnAFP promoter from a polar diatom, which is amplified using PCR or inverse PCR. Following this, bioinformatic analysis is performed using tools such as PLACE and Softberry to identify key cis-acting regulatory elements, including low-temperature response elements (LTREs) and other stress-responsive motifs. These promoters sequences are then cloned upstream of reporter genes such as Gaussia luciferase (GLuc) or mVenus, and introduce them into a host organism via electroporation. The successful transformants are usually selected using an antibiotic resistance marker. Further, the functional validation of promoters inducibility is confirmed by exposing engineered strains to specific stimuli (e.g., changing the temperature from 25 °C to 10 °C) and quantifying the subsequent response using quantitative real-time PCR or reporter protein activity assays. Finally, the promoter is refined using successive truncation experiments to establish the minimal functional sequence length and identify specific regulatory motifs, such as SEF4-BS-like regions, required for the induction response (Schroda et al., 2000a). Complementary transcriptomic approach (RNA-seq) and quantitative PCR (qRT-PCR) are frequently used to identify candidate inducible promoters by identifying genes with condition specific or growth phase dependent expression patterns. For example, genes showed increased expression during the late exponential phase in development, have been used to create inducible promoter such as Pcrt, associated with calreticulin gene. Further the bioinformatic analysis helps in identifying a target gene, such as calreticulin, where an approx. A ∼1.5 kb upstream region was identified, containing crucial regulatory elements, including TATA boxes and bZIP-binding sites. These identified regions are then incorporated into a species-specific expression vector containing a codon-optimized reporter gene. Such promoters can be activated by diverse abiotic stressors such as nitrogen (N) starvation (24 h), heat stress (42 °C for 1 h), and endoplasmic reticulum (ER) stress (Lee et al., 2020).

Similarly, salt-inducible promoters (SIPs) have been identified from the freshwater microalgae Chlorella vulgaris PKVL7422 by searching for genes that are elevated after salt treatment. In a transcriptomic study, when C. vulgaris was treated with salt, genes involved in the synthesis of methyl jasmonic acid (MeJA), gibberellin (GA), and abscisic acid (ABA) were found to be upregulated. These promoters often reported to contain several stress-responsive cis-acting elements, which are functionally tested by cloning them with a reporter gene (e.g., Gluc) and by assigning expression in salt treatment. Additionally, treating the transformed C. vulgaris with salt and MeJA, GA, and ABA increased the expression level of recombinant luciferase (Kim et al., 2023). Collectively, inducible promoter systems operate through well-defined molecular mechanisms that involve ligand- or stimulus-dependent modulation of transcription factors. Figure 2 illustrates the positive and negative inducible promoter expression systems and the molecular mechanisms, in which the inducer activates transcription by converting inactive TFs into recruiters or releasing repressors from operators. This flexibility allows precise, condition-specific control of gene expression.

3.2 Synthetic inducible promoters

In contrast to native promoters the synthetic promoters are non-natural DNA regulatory sequence designed to reliably drive the expression of a target gene. Structurally, they consist of a core region with DNA elements to recruit basic transcriptional machinery and an upstream proximal region with targets for additional transcription factors (TFs). Unlike native promoters, the synthetic promoter can be assembled artificially or from pre-existing parts in a non-natural way. The key advantage over native promoters is that they can be designed with minimal footprints, reducing the size of the expression cassette. This simplifies Synthetic biology applications, such as de novo metabolic pathway engineering. The synthetic promoters can be generated using two distinct strategies: top-down and bottom-up techniques. Top-down design utilizes well-defined native promoters as templates and modifies them accordingly. Bottom-up promoter design builds promoters from scratch, resulting in new assemblies (Milito, 2023). Synthetic inducible promoters often integrate a strong native promoter element, such as βTUB2, with HSP70A to boost basal activity and resistance to silencing. Post-transcriptional performance can also be enhanced by inserting introns into the 5′UTR and by combinatorially integrating specific cis-motifs (such as CCCATGCA and ATANTT) at specific locations. Such engineering can yield dramatic gains; for example, the designed AβSAP(i) promoter increases the synthesis of high-value bioproducts, including terpenes, by 18 times compared to its native counterpart and by 4 times compared to commonly used promoter systems (Einhaus et al., 2021a). In addition to promoter engineering, synthetic TFs can be designed by combining specialized DNA-binding domains with powerful activators or repressors to control the expression of hybrid promoters designed to contain the target DNA sequence. A prominent example is the Tet-off system, in which aVP16 transcriptional activator is fused with a Tet repressor to form a chimeric transcriptional activator (tTA), which binds to tandem Tet operators to drive transcription in the absence of tetracycline. The presence of tetracycline prevents the repressor from binding the operator, thereby inhibiting the transcription. Meanwhile, in the Tet-On system, the response is reversed by utilizing a Tet repressor (TetR), which only binds to the Tet operator in the presence of tetracycline, resulting in inducible activation of transcription (Xiao et al., 2013; Hacker and Balasubramanian, 2016).

4 Molecular basis of a promoter system

Promoters are cis-regulatory DNA sequences that recruit the transcriptional machinery, including RNA polymerase (RNAP) and associated factors, to initiate gene expression. In eukaryotes, multiple RNAP complexes are present in the nucleus and DNA-containing organelles, whereas bacteria and archaea rely on a single RNAP. Although numerous factors influence gene expression, the promoter remains the primary driver of transcriptional initiation. Therefore, the cellular engineering strategies frequently target promoter regions to modulate gene expression. Extensive research has focused on characterizing, optimizing, and redesigning promoter sequences to fine-tune transcriptional output. As a result, promoter engineering is increasingly recognized as an important field of synthetic biology (Cazier and Blazeck, 2021a). The term promoter engineering refers to the process of developing novel, cis-acting DNA elements that control transcription and differ from natural promoters. Synthetic promoters can have stronger expression, shorter length, fewer repetitive sequences, graded expression, or unique inducibility to support downstream cellular and metabolic engineering applications. For example, strengthening a promoter boosts enzyme levels beyond native limits, thereby increasing metabolic flow through pathways for biofuels.

In Prokaryotic systems, the promoter’s architecture is relatively well defined and primarily relies on conserved consensus sequences recognized by RNA polymerase and its associated sigma factors. For example, in Escherichia coli, the key regulatory elements include the −10 and −35 regions, which are important for promoter recognition and transcription initiation. Whereas the eukaryotic promoters exhibit greater structural and functional complexity, comprising modular regulatory regions that allow fine-tuned control of gene expression. These include the core, proximal promoter, and distal regulatory regions. The core promoter is located near the transcription start site (TSS) and contains motifs required for pre-initiation complex assembly, such as the TATA box, BRE (TFIIB recognition element), Inr (initiator element), and Y patch. Upstream of this lies the Proximal promoter region, which is enriched in transcription factor binding sites (TFBS) that control the gene’s responsiveness to environmental and developmental signals. The distal regulatory region consists of enhancers, silencers, or insulators and is located several kilobases away from the transcription start site. This regulates the central promoter via long-range regulatory interactions mediated by the DNA looping mechanism (Cazier and Blazeck, 2021a; Cardiff et al., 2024; Yaschenko et al., 2026). Figure 3 presents a schematic of the promoter architecture and transcriptional regulation, showing distal and proximal cis-regulatory elements, transcription factors, the core promoter region containing the TATA box and transcription start site, and the assembly of the preinitiation complex with RNA pol II to drive expression of the gene of interest. Promoters can be engineered to respond to specific biological contexts, thereby promoting gene expression in response to defined environmental or intracellular cues, such as the concentration of an exogenous small molecule (an inducer), cell temperature, oxygen availability, or fluctuations in intracellular metabolites. This inducibility is primarily mediated by condition-dependent transcription factors. Inducible promoters contain TFBSs and hence can activate complex genomic circuits only under specific conditions, making them excellent for metabolic and cellular engineering applications. These features make them highly valuable tools in metabolic engineering and synthetic biology for achieving specific control over gene expression. Before porting functionality into a new hybrid promoter scaffold, it is often necessary to elucidate the TFBSs within an upstream activating sequence (UAS) that are responsible for condition-specific gene expression, typically by experiments that truncate the UAS or mutagenize putative TFBS sites. Interestingly, after the discovery of TF-TFBS pairings, they can either be used directly in their original host or introduced concurrently into a new organism to establish non-native mechanisms of inducible gene control.

The potential of microalgae as a sustainable and affordable biofuel feedstock may be fully realized through synthetic biology, which involves the building of new biological systems or the redesign of existing ones. The lipid content and productivity of microalgae are among the main goals for advancements driven by synthetic biology. However, this high lipid content often results in a trade-off between quantity and quality, reducing overall biomass production. Synthetic biology techniques can create engineered microalgal strains that maintain high lipid yields while increasing overall biomass production through targeted modifications, such as introducing or upregulating key genes involved in lipid biosynthesis and carbon partitioning. Such strategies can help address current obstacles that limit the commercialization of algal biofuels. Recent advances have established a comprehensive genetic toolkit for microalgae engineering. The key components include synthetic promoters, inducible gene switches, and precise genome-editing tools such as CRISPR-Cas9 systems, transcription activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs) (Makepa and Chihobo, 2025). These tools allow researchers to enhance the production of target substances (such as lipids) without creating metabolic overload or cellular stress. Collectively, advances in synthetic biology are helping unlock the full potential of microalgae for the production of next-generation biofuels.

5 Integrating inducible promoter engineering with a two-step cultivation system

As already discussed, the endogenous promoter concept and the limitation of the two-step cultivation system, where the constitutive expression system is not always expressed in a controlled manner, and induction in stage II can lead to off-target protein production. To overcome these effects, while maintaining controlled induction with minimal metabolic loss in stage II, an effective alternative expression system is needed. One way to overcome the limitations of the conventional two-stage cultivation system is to integrate an inducible promoter system to precisely regulate gene expression during the production phase (Stage II). In this process, microalgae cultures are initially propagated under optimal growth conditions to get higher biomass (stage I), followed by inducing the gene of interest in the second stage with an appropriate chemical or environmental inducer. Exogenous small molecules (inducers), cell temperature, and oxygen availability are used. Transcription factors that only bind to their corresponding binding sites under certain conditions are the driving force behind this inducibility. This will enable induction by the stress factor, with a controlled metabolic flux directed towards a targeted pathway. Inducible promoters contain such TFBSs and can activate complex genomic circuits only under specific conditions, making them excellent for metabolic and cellular engineering applications (Dahl et al., 2013; Ede et al., 2016). This expression system ideally has three characteristics: induction is extremely selective rather than “leaky,” and it is tunable. And in the case of a chemical inducer, affordability and non-toxicity are advantageous characteristics. Even though it has advantages, care should be taken before porting functionality into a new hybrid promoter scaffold; it is often necessary to identify the TFBSs within a UAS responsible for condition-specific induction of gene expression, typically by truncating the UAS or mutagenizing putative TFBS sites. Interestingly, after the discovery of TF-TFBS pairings, they can either be used directly in their original host or introduced concurrently into a new organism to establish nonnative mechanisms of inducible gene control (Åkerfelt et al., 2010; Schödel et al., 2011; Weinhandl et al., 2014; Wang, 2023). To aid understanding, Figure 4 presents a diagrammatic representation of the hypothesis for an induced two–step cultivation system in microalgae.

6 Checkpoints for the production of lipids from microalgae systems

Microalgae are an extremely promising platform for long-term CO2 mitigation and the synthesis of renewable lipids. Triacylglycerol (TAG), a dense energy-storage molecule that makes up between 60 and 70 percent of the dry mass of algal cells (Sharma et al., 2018)It is the primary means by which microalgae produce biofuel. Photosynthesis, carbon fixation, fatty acid synthesis, and TAG assembly are the initial steps in TAG formation. The foundation of TAG synthesis is the fatty acid production pathway, which begins with acetyl-CoA carboxylase, which converts acetyl-CoA to malonyl-CoA, and acetyl-CoA synthase, which produces acetyl-CoA precursors. Then, genes like glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidic acid acyltransferase (LPAAT), phosphatidic acid phosphatase (PAP), diacylglycerol acyltransferase (DGAT), and phospholipid: PDAT catalyze the sequential incorporation of fatty acids onto a glycerol backbone during TAG assembly. Other routes to maximize lipid synthesis include engineering competing pathways and carbon partitioning. That involves blocking the starch synthesis pathway, suppressing lipid catabolism, diverting glycolytic flux, and altering transcriptional regulation (Sharma et al., 2018). Significant progress has been made in understanding the molecular mechanisms driving TAG synthesis, storage, and turnover. Several key transcription factors, such as NRR1, PSR1, TAR1, and MYB1, have been identified to play important roles in regulating lipid accumulation under nutrient-limited conditions by controlling genes involved in fatty acid metabolism (Jin et al., 2015; Banerjee et al., 2016; Zhang et al., 2025). Table 1 lists lipid biosynthesis genes reported in various microalgae, which can serve as target genes for promoter engineering. These genes can be promoter-engineered and induced in phase II of the cultivation system. This approach may facilitate the enhanced lipid accumulation while minimizing metabolic burden during the growth phase, thereby improving overall process efficiency.

TABLE 1. Genes reported so far in microalgae for enhanced lipid synthesis.

7 Challenges in the inducible promoter for the two-step cultivation system

The intrinsic complexity of genetic control and technological constraints in experimental and computational techniques are two of the major obstacles to promoter engineering. Promoter functions are controlled by a multi-layered regulatory architecture that is not entirely understood, particularly in eukaryotes. For example, a substantial proportion of genes in yeast and humans lack classical components such as the TATA box or the initiator (Inr) motif, yet remain transcriptionally competent. Furthermore, the particular “scaffold” or surrounding DNA sequence of the promoter frequently determines its strength and regulatory responsiveness. Additionally, the location, whether on an episomal plasmid or integrated into the genome, as well as the particular development conditions of the cell, affect its function. Distal enhancers are found thousands of base pairs away in mammals and higher eukaryotes; because of their remote location and intricate interactions, they are more difficult to construct than proximal promoter components (Cazier and Blazeck, 2021b). Engineered promoters are frequently influenced by numerous unintended regulatory signals, reflecting the sensitivity of transcriptional systems. For example, depending on the terminator employed, the RPL23 promoter showed significant differences in transformation efficiency. Such observations highlight the challenges of achieving predictable promoter behavior. The key objective of promoter engineering is to develop regulatory components that consistently control transgene expression. This capability represents a critical prerequisite for in the management of synthetic genetic circuits. However, many genetic components lack consistency in non-model organism including microalgae, as their standardization was achieved only in established hosts, such as yeast or bacteria, making it challenging to achieve precise control over gene expression dynamics (Einhaus et al., 2021b). The use of inducible promoters further explains these challenges. For example, a well-known inducible expression tool, the HSP70A promoter, is widely used in microalgal engineering for its ability to enhance basal transcription and alleviate transgene gene silencing. While advantageous in certain contexts, this property leads to a fundamental trade-off in inducible systems. Specifically, in an inducible system where the gene must be completely “off” until activated, high basal activity can lead to transcriptional leakiness, which is undesirable when stringent repression in the uninduced state is required. On the other hand, in the absence of such expression-enhancing components, the transgene might not express due to highly efficient DNA silencing mechanisms (Schroda et al., 2000b).”

Promoter engineering in microalgae has other challenges, including a limited regulatory toolkit and an underdeveloped genetic toolkit. Even for well-researched model organisms like Chlamydomonas reinhardtii, a systematic understanding of regulatory factors remains incomplete. The current engineering strategies rely heavily on an empirical, low-throughput approach rather than on rational design or predictive modeling. Further, forecasting the promoter function and regulatory behavior is very challenging due to the intrinsic complexity of microalgal biological systems. In particular, heterologous genetic elements often perform poorly as regulatory components, as components derived from other species fail to function efficiently in microalgae due to differences in transcriptional machinery. Additionally, the interplay between Promoter-transcriptional terminator can act as an important determinant of gene expression level. However, the synergy between promoters and terminators remains insufficiently investigated, despite evidence that a specific promoter-terminator combination can significantly affect transcriptional output (Gan et al., 2025). Moreover, limited Species accessibility is another bottleneck in determining the transformation’s efficiency, as only very few algae species have undergone routine transformation, leaving many industrially important strains genetically intractable (Gan et al., 2025). Even in transformable species, challenges associated with gene delivery, genomic integration, and long-term transgene stability persist. Further, in several microalgal strains, including Nannochloropsis spp., methods such as homologous recombination and CRISPR/Cas9 exhibit comparatively low efficacy. Moreover, stable transformation in microalgae becomes more difficult because they possess highly effective DNA silencing mechanisms that can inhibit the expression of supplied transgenes (Crozet et al., 2018; Gimpel, 2015).

8 Conclusion

This review concludes that the two-step cultivation strategy has emerged as a practical framework for microalgal bioprocessing; however, the inherent trade-off between biomass growth and metabolite synthesis currently prevents them from being commercially viable. Because the conventional single-stage cultivation is suboptimal for simultaneous growth and product yield, where stress conditions needed for lipid induction usually prevent cell growth, and is still constrained by poor temporal control of stress responses, variable induction strength, and strain-dependent differences in productivity. Whereas the two-step cultivation approach, which distinguishes between focused induction and optimal growth (Stage I), has emerged as an effective strategy to partially address this limitation. However, this approach remains limited by poor control over stress responses, resulting in suboptimal productivity. In this context, dynamic switch-based metabolic regulation offers a key means of overcoming these constraints. The integration of inducible genetic switches, such as stress-responsive or synthetic promoters, enables precise temporal control over metabolic pathways. Further, by restricting metabolite synthesis to the post-growth phase, both a large amount of biomass and the desired metabolite can be produced. Collectively, advances in the two-step cultivation system using synthetic biology can help unlock the potential of microalgae for the commercial production of microalgal biofuels. Future studies should concentrate on developing robust host strains and scalable cultivation platforms, as well as enhancing circuit stability, induction specificity, and promoter tightness. Additionally, research is required to integrate a two-stage culture system with omics-guided target selection, genome editing, and process optimization. All things considered, combining a two-step culture with a synthetic biology approach offers a potent way to overcome current obstacles and advance the production of improved microalgal biofuel toward commercial viability.

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Footnotes

Edited by: Changliang Nie, Qingdao University, China

Reviewed by: Denzel Christopher Makepa, Chinhoyi University of Technology, Zimbabwe

Fariha Kanwal, Lahore College for Women University, Pakistan