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Microalgae hold enormous potential to provide a safe and sustainable source of high-value compounds, acting as carbon-fixing biofactories that could help to mitigate rapidly progressing climate change. Bioengineering microalgal strains will be key to optimizing and modifying their metabolic outputs, and to render them competitive with established industrial biotechnology hosts, such as bacteria or yeast. To achieve this, precise and tuneable control over transgene expression will be essential, which would require the development and rational design of synthetic promoters as a key strategy. Among green microalgae, Chlamydomonas reinhardtii represents the reference species for bioengineering and synthetic biology; however, the repertoire of functional synthetic promoters for this species, and for microalgae generally, is limited in comparison to other commercial chassis, emphasizing the need to expand the current microalgal gene expression toolbox. Here, we discuss state-of-the-art promoter analyses, and highlight areas of research required to advance synthetic promoter development in C. reinhardtii. In particular, we exemplify high-throughput studies performed in other model systems that could be applicable to microalgae, and propose novel approaches to interrogating algal promoters. We lastly outline the major limitations hindering microalgal promoter development, while providing novel suggestions and perspectives for how to overcome them.

Plant synthetic biology allows for the reprogramming of biological systems to promote sustainable agriculture. It has a range of applications, including improving nutritional quality, engineering resilient crops, creating new materials, and producing various bioproducts. In addition, plant synthetic biology provides a new way to answer fundamental questions in plant biology. This Special Issue presents a collection of review articles that delve into the current and future state of plant synthetic biology.

Flavonoids are a group of secondary metabolites from plants that have received attention as high value-added pharmacological substances. Recently, a robust and efficient bioprocess using recombinant microbes has emerged as a promising approach to supply flavonoids. In the flavonoid biosynthetic pathway, the rate of chalcone synthesis, the first committed step, is a major bottleneck. However, chalcone synthase (CHS) engineering was difficult because of high-level conservation and the absence of effective screening tools, which are limited to overexpression or homolog-based combinatorial strategies. Furthermore, it is necessary to precisely regulate the metabolic flux for the optimum availability of malonyl-CoA, a substrate of chalcone synthesis. In this study, we engineered CHS and optimized malonyl-CoA availability to establish a platform strain for naringenin production, a key molecular scaffold for various flavonoids. First, we engineered CHS through synthetic riboswitch-based high-throughput screening of rationally designed mutant libraries. Consequently, the catalytic efficiency (kcat/Km) of the optimized CHS enzyme was 62% higher than that of the wild-type enzyme. In addition to CHS engineering, we designed genetic circuits using transcriptional repressors to fine-tune the malonyl-CoA availability. The best mutant with synergistic effects of the engineered CHS and the optimized genetic circuit produced 98.71 mg/L naringenin (12.57 mg naringenin/g glycerol), which is the highest naringenin concentration and yield from glycerol in similar culture conditions reported to date, a 2.5-fold increase compared to the parental strain. Overall, this study provides an effective strategy for efficient production of flavonoids.

Microbial consortia have been considered potential platforms for bioprocessing applications. However, the complexity in process control owing to the use of multiple strains necessitates the use of an efficient population control strategy. Herein, we report circuit-guided synthetic acclimation as a strategy to improve biochemical production by a microbial consortium. We designed a consortium comprising alginate-utilizing Vibrio sp. dhg and 3-hydroxypropionic acid (3-HP)-producing Escherichia coli strains for the direct conversion of alginate to 3-HP. We introduced a genetic circuit, named “Population guider”, in the E. coli strain, which degrades ampicillin only when 3-HP is produced. In the presence of ampicillin as a selection pressure, the consortium was successfully acclimated for increased 3-HP production by 4.3-fold compared to that by a simple co-culturing consortium during a 48-h fermentation. We believe this concept is a useful strategy for the development of robust consortium-based bioprocesses.

Physical compartmentalization of metabolism using membranous organelles in eukaryotes is helpful for chemical biosynthesis to ensure the availability of substrates from competitive metabolic reactions. Bacterial hosts lack such a membranous system, which is one of the major limitations for efficient metabolic engineering. Here, we employ kinetic compartmentalization with the introduction of an unnatural enzymatic reaction by an engineered enzyme as an alternative strategy to enable substrate availability from competitive reactions through kinetic isolation of metabolic pathways. As a proof of concept, we kinetically isolate the itaconate synthetic pathway from the tricarboxylic acid cycle in Escherichia coli, which is natively separated by mitochondrial membranes in Aspergillus terreus. Specifically, 2-methylcitrate dehydratase is engineered to alternatively catalyze citrate and kinetically secure cis-aconitate for efficient production using a high-throughput screening system. Itaconate production can be significantly improved with kinetic compartmentalization and its strategy has the potential to be widely applicable.

Proper carbon flux distribution between cell growth and production of a target compound is important for biochemical production because improper flux reallocation inhibits cell growth, thus adversely affecting production yield. Here, using a synthetic biosensor to couple production of a specific metabolite with cell growth, we spontaneously evolve cells under the selective condition toward the acquisition of genotypes that optimally reallocate cellular resources. Using 3-hydroxypropionic acid (3-HP) production from glycerol in Escherichia coli as a model system, we determine that mutations in the conserved regions of proteins involved in global transcriptional regulation alter the expression of several genes associated with central carbon metabolism. These changes rewire central carbon flux toward the 3-HP production pathway, increasing 3-HP yield and reducing acetate accumulation by alleviating overflow metabolism. Our study provides a perspective on adaptive laboratory evolution (ALE) using synthetic biosensors, thereby supporting future efforts in metabolic pathway optimization.

RNA-binding proteins (RBPs) are crucial factors of post-transcriptional gene regulation and their modes of action are intensely investigated. At the center of attention are RNA motifs that guide where RBPs bind. However, sequence motifs are often poor predictors of RBP-RNA interactions in vivo. It is hence believed that many RBPs recognize RNAs as complexes, to increase specificity and regulatory possibilities. To probe the potential for complex formation among RBPs, we assembled a library of 978 mammalian RBPs and used rec-Y2H matrix screening to detect direct interactions between RBPs, sampling > 600 K interactions. We discovered 1994 new interactions and demonstrate that interacting RBPs bind RNAs adjacently in vivo. We further find that the mRNA binding region and motif preferences of RBPs deviate, depending on their adjacently binding interaction partners. Finally, we reveal novel RBP interaction networks among major RNA processing steps and show that splicing impairing RBP mutations observed in cancer rewire spliceosomal interaction networks. The dataset we provide will be a valuable resource for understanding the combinatorial interactions of RBPs with RNAs and the resulting regulatory outcomes.

The identification of each cell type is essential for understanding multicellular communities. Antibodies set as biomarkers have been the main toolbox for cell-type recognition, and chemical probes are emerging surrogates. Herein we report the first small-molecule probe, CDgB, to discriminate B lymphocytes from T lymphocytes, which was previously impossible without the help of antibodies. Through the study of the origin of cell specificity, we discovered an unexpected novel mechanism of membrane-oriented live-cell distinction. B cells maintain higher flexibility in their cell membrane than T cells and accumulate the lipid-like probe CDgB more preferably. Because B and T cells share common ancestors, we tracked the cell membrane changes of the progenitor cells and disclosed the dynamic reorganization of the membrane properties over the lymphocyte differentiation progress. This study casts an orthogonal strategy for the small-molecule cell identifier and enriches the toolbox for live-cell distinction from complex cell communities.

RNA splicing is widely dysregulated in cancer, frequently due to altered expression or activity of splicing factors (SFs). Microexons are extremely small exons (3–27 nucleotides long) that are highly evolutionarily conserved and play critical roles in promoting neuronal differentiation and development. Inclusion of microexons in mRNA transcripts is mediated by the SF Serine/Arginine Repetitive Matrix 4 (SRRM4), whose expression is largely restricted to neural tissues. However, microexons have been largely overlooked in prior analyses of splicing in cancer, as their small size necessitates specialized computational approaches for their detection. Here, we demonstrate that despite having low expression in normal nonneural tissues, SRRM4 is further silenced in tumors, resulting in the suppression of normal microexon inclusion. Remarkably, SRRM4 is the most consistently silenced SF across all tumor types analyzed, implying a general advantage of microexon down-regulation in cancer independent of its tissue of origin. We show that this silencing is favorable for tumor growth, as decreased SRRM4 expression in tumors is correlated with an increase in mitotic gene expression, and up-regulation of SRRM4 in cancer cell lines dose-dependently inhibits proliferation in vitro and in a mouse xenograft model. Further, this proliferation inhibition is accompanied by induction of neural-like expression and splicing patterns in cancer cells, suggesting that SRRM4 expression shifts the cell state away from proliferation and toward differentiation. We therefore conclude that SRRM4 acts as a proliferation brake, and tumors gain a selective advantage by cutting off this brake.

Although brown macroalgae holds potential as an alternative feedstock, its utilization by conventional microbial platforms has been limited due to the inability to metabolize one of the principal sugars, alginate. Here, we isolate Vibrio sp. dhg, a fast-growing bacterium that can efficiently assimilate alginate. Based on systematic characterization of the genomic information of Vibrio sp. dhg, we establish a genetic toolbox for its engineering. We also demonstrate its ability to rapidly produce ethanol, 2,3-butanediol, and lycopene from brown macroalgae sugar mixture with high productivities and yields. Collectively, Vibrio sp. dhg can be used as a platform for the efficient conversion of brown macroalgae sugars into diverse value-added biochemicals

Ribozymes are functional RNA molecules that can catalyze biochemical reactions. Since the discovery of the first catalytic RNA, various functional ribozymes (e.g., self-cleaving ribozymes, splicing ribozymes, RNase P, etc.) have been uncovered, and their structures and mechanisms have been identified. Ribozymes have the advantage of possessing features of “RNA” molecules; hence, they are highly applicable for manipulating various biological systems. To fully employ ribozymes in a broad range of biological applications in synthetic biology, a variety of ribozymes have been developed and engineered. Here, we summarize the main features of ribozymes and the methods used for engineering their functions. We also describe the past and recent efforts towards exploiting ribozymes for effective and novel applications in synthetic biology. Based on studies on their significance in biological applications till date, ribozymes are expected to advance technologies in artificial biological systems.

Understanding which proteins and RNAs directly interact is crucial for revealing cellular mechanisms of gene regulation. Efficient methods allowing to detect RNA-protein interactions and dissect the underlying molecular origin for RNA-binding protein (RBP) specificity are in high demand. The recently developed recombination-Y3H screening (rec-Y3H) enabled many-by-many detection of interactions between pools of proteins and RNA fragments for the first time. Here, we test different conditions for protein-RNA interaction selection during rec-Y3H screening and provide information on the screen performance in several selection media. We further show that rec-Y3H can detect the nucleotide and amino acid sequence determinants of protein-RNA interactions by mutating residues of interacting proteins and RNAs simultaneously. We envision that systematic RNA-protein interface mutation screening will be useful to understand the molecular origin of RBP selectivity and to engineer RBPs with targeted specificities in the future.