Metabolic Engineering Communications

BackgroundSuccinic acid (SA) is a four-carbon dicarboxylic acid of considerable industrial relevance, with applications spanning the food, chemical, and pharmaceutical sectors. The remarkable acid tolerance of the yeast Yarrowia lipolytica makes it a promising microbial cell factory for SA production. Numerous metabolic engineering strategies have focused on disrupting genes encoding the succinate dehydrogenase (SDH) complex to enhance SA accumulation. However, such a modification is associated with impaired growth and the accumulation of by-products, notably acetic acid (AA). ResultsTo improve growth capacity, SA productivity, and reduce AA formation in Y. lipolytica SDH5-deficient strains (Sdh5{Delta}), carbon flux from glycolysis was partially redirected toward the pentose phosphate pathway by overexpression of the native genes encoding glucose-6-phosphate dehydrogenase (ZWF1) and 6-phosphogluconate dehydrogenase (GND1), thereby enhancing NADPH generation. The resulting strain was further engineered to increase NADH availability for the mitochondrial electron transport chain by overexpressing genes encoding either a mutated NADPH-dependent malate dehydrogenase (TfMdh) from Thermus flavus or the soluble transhydrogenase (EcSthA) from Escherichia coli, enabling indirect conversion of NADPH to NADH. This strategy resulted in 2-fold and 2.2-fold increase in SA productivity and titre, respectively, compared to the Sdh5{Delta}-ALE strain during bioreactor cultivation on glucose-based media. Moreover, AA accumulation was reduced 1.2-fold, while growth rates were significantly improved. ConclusionsThe proposed engineering strategies, especially heterologous expression of EcSthA, partly alleviated energy limitations in Y. lipolytica Sdh5{Delta} strain, resulting in improved SA productivity and growth performance.

Engineering heterologous utilization of substrates requires selection of catabolic pathways that balance strain performance and product biosynthesis. Here, we compare the oxidative and isomerase arabinose utilization pathways in Pseudomonas putida strains engineered for cis,cis-muconic acid production from glucose and xylose. Based on the point of entry into central carbon metabolism, we hypothesized that the oxidative arabinose pathway would enable higher productivity while the arabinose isomerase pathway would enable higher muconate yield. In both strains, additional modifications were engineered to improve muconic acid production including sugar transporter tuning, catechol 1,2-dioxygenase overexpression, a feedback-resistant DAHP synthase, and a flux-stabilizing gltA variant. Consistent with our hypothesis, the oxidative arabinose pathway supported faster growth and higher productivity (0.58 g/L/h), whereas the arabinose isomerase pathway improved carbon efficiency, achieving muconate yields of up to 50 C-mol% in fed-batch bioreactors. Process modeling indicates that these performance metrics can reduce the minimum selling price of muconate-derived adipic acid to $2.74/kg and greenhouse gas emissions to 1.31 kg CO2e/kg, approaching cost parity and reducing emissions by 86% relative to fossil carbon-derived adipic acid. Overall, this study presents a systematic comparison of sugar catabolic pathways that enabled development of strains suited for the tradeoffs between rate and yield.

Cyanobacterial natural products are a rich source of bioactive compounds, yet their heterologous production remains challenging. This study investigates the feasibility of expressing the lyngbyatoxin A (LTXA) biosynthetic gene cluster in a fungal host. The lyngbyatoxin biosynthetic genes (ltxA, ltxB, ltxC) were individually cloned and expressed in Aspergillus oryzae NSAR1 under the control of an inducible promoter. Metabolite production was assessed using LC- MS, and transcriptional analysis was performed by RT-PCR. Codon-optimized constructs and precursor feeding experiments were employed to evaluate pathway functionality. No production of LTXA or pathway intermediates was detected upon co-expression of ltxA-C despite confirmed transcription of ltxB and ltxC. RT-PCR analysis revealed truncation of the ltxA transcript, suggesting incompatibility with fungal transcriptional or splicing machinery. In contrast, expression of a codon-optimized ltxC enabled biotransformation of indolactam V to LTXA in A. oryzae, confirming functional expression of the prenyltransferase. These results highlight transcriptional limitations as a key barrier to heterologous expression of cyanobacterial NRPS pathways in fungal hosts, while demonstrating that downstream tailoring enzymes can remain functional. This work provides insights for future engineering of fungal platforms for cyanobacterial natural product biosynthesis.

Budding yeast Saccharomyces cerevisiae is a workhorse chassis for producing added food and agricultural compounds. However, building multi-enzymatic pathways for these chemicals often requires iterative genomic integration, underscoring the need for efficient, rapid genome-editing tools that can reliably target transcriptionally active chromosomal regions. In this study, to accelerate strain construction, we established a genome-editing toolkit to rapidly engineer eight loci, highly expressed hot-spots, but nonessential genomic sites suitable for stable pathway assembly. Our approach integrates three key design features: (i) selectable markers to enable rapid screening of edited cells, (ii) extended homology arms that leverage the yeast homology-directed repair machinery for robust genomic integration, and (iii) co-delivery of Cas9 and guide RNAs to promote efficient double-stranded DNA breaks at specific integration sites. The sequence independence of FASTOP relies on the release of integration cassettes from integrative vectors, mediated by restriction digestion at two flanking multiple-cutting sites in the integration module to minimize the risk of introducing sequence errors during PCR amplification of the integration cassettes. Following the introduction of a fluorescent reporter cassette, we observed high integration efficiencies across the target sites. We then integrated the biosynthetic pathway of plant-derived flavonoid naringenin into the hot-spots of the yeast genome using the FASTOP toolkit. Our results demonstrated that upon expressing the five essential genes in simple shake flask culture, naringenin production reached 505.7 mg/L, representing a significant (69-fold) increase over previously reported titers for comparable minimal heterologous pathways in S. cerevisiae. Together, the FATSOP toolkit provides a user-friendly platform for reliably modifying hot-spot loci to rapidly construct multi-enzymatic metabolic pathways in S. cerevisiae, while achieving high production levels for high-value food-relevant metabolites.

Muconic acid is an industrially valuable molecule that can be biologically produced from diverse biogenic and waste-derived feedstocks, including sugars and lignin- and plastic-derived aromatic compounds. However, accumulation of protocatechuate (PCA) has been observed in multiple microbes engineered for muconate production when the PCA decarboxylase, AroY, is used. This raises the question of whether PCA decarboxylation represents a rate-limiting step and how this bottleneck might be alleviated, especially given the toxicity and reactivity of PCA and catechol intermediates. To address this, we performed adaptive laboratory evolution (ALE) on a strain of Pseudomonas putida originally engineered for muconate production from aromatic compounds, but with catBC restored, to select for improved conversion of PCA and, in separate lineages, 4-hydroxybenzoate. Contrary to our expectations, the predominant beneficial mutations localized to the catA1 cassette encoding catechol 1,2-dioxygenase, rather than aroY or its associated cofactor biosynthesis genes. Transcriptomic analysis revealed elevated catA1 expression in evolved isolates from ALE, and introduction of these mutations improved productivity in strains designed for muconate production from both aromatic and sugar substrates. Quantitative proteomics and biochemical assays demonstrated that the mutations also led to increased CatA1 protein abundance and modest enhancements in catalytic efficiency, respectively, with strain phenotypes largely driven by high CatA1 levels and potentially synergistic kinetic improvements. Additional reverse-engineering studies identified variants with modest effects on muconate accumulation, including those with potential to enhance biosynthesis of the prenylated FMN cofactor of AroY. Collectively, these results indicate that catechol, not PCA, is the principal bottleneck in muconate production via the PCA decarboxylation route originally demonstrated by Draths et al., refining our understanding of pathway limitations and offering new strategies for improving rate, yield, and strain resilience in muconate bioproduction. HighlightsO_LIAccumulation of metabolic intermediates was alleviated by adaptive laboratory evolution C_LIO_LISequencing, proteomics, and enzyme kinetics revealed mechanisms for adaptation C_LIO_LIIncreased CatA1 expression reduced bottlenecks and improved muconate production C_LI

Robust strain stability is essential for industrial-scale fatty alcohol production, as metabolic burden and toxicity not only constrain productivity but also create selective pressure for low-producing or non-producing subpopulations. This genetic instability leads to genetic heterogeneity and compromises strain performance during large-scale fermentation. In this study, we investigated the production stability of fatty alcohol-producing Yarrowia lipolytica strains and developed systematic strategies to improve the genetic stability of engineered strains for scale-up production. In a mock fermentation scale-up, a fatty acyl-CoA reductase (FAR)-expressing strain lost fatty alcohol production after five consecutive passages. To address this, we fused FAR to phosphoglycerate kinase I (PGK1), a gene essential to cell growth, to promote the stability of FAR expression and prevent production loss. This strategy extended fatty alcohol production by one passage. Additionally, FAR was fused to GFP and extended production stability by four additional passages. In parallel, competitive co-culture experiments, in which producing strains were cultured alongside non-producing mutants, revealed that when non-producers emerged with a frequency of 10-5, they dominated the fermentation population within six passages; at 10%, they took only two passages. Furthermore, deep sequencing of strains that demonstrate different levels of stability and fatty alcohol productivity identified mutation patterns that contribute to strain instability. These findings provide insights into engineering Y. lipolytica with enhanced genetic stability for scale-up fatty alcohol production.

Wickerhamomyces ciferrii is a non-model diploid yeast that naturally produces tetraacetyl phytosphingosine (TAPS), a sphingoid base used in cosmetic and dermatological applications. However, its strong preference for non-homologous end joining (NHEJ) over homologous recombination (HR) limits conventional genome editing, while disruption of LIG4, a core NHEJ gene, compromises cellular fitness. Here, we repurposed native NHEJ activity to develop a homology-independent multicopy genome integration platform for W. ciferrii. The platform combines three optimized donor-design features: telomeric end-shielding with two tandem copies of an 11 bp repeat to improve linear donor persistence, a defective URA5 auxotrophic marker to enrich multicopy integrants, and 5'-phosphorylated donor termini to enhance transformant recovery and integration output. These features were consolidated into the platform vector pTdmVU5. As a metabolic engineering demonstration, multicopy integration of LCB1 and LCB2, encoding the two subunits of serine palmitoyltransferase, increased TAPS titer by 2.7-fold. This work converts the native NHEJ bias of W. ciferrii from a barrier to precise genome editing into a practical tool for pathway amplification and establishes a framework for engineering NHEJ-dominant non-model yeasts.

Monoterpene Indole Alkaloids (MIAs) are a diverse family of plant natural products with various medicinal applications. Although MIAs, such as vinblastine and reserpine, are clinically validated, sourcing of MIAs for clinical use or drug discovery from natural resources or via chemical synthesis is hampered due to their scarcity and chemical complexity. Refactoring MIA biosynthesis pathways in microbial cell factories could offer an alternative, more stable and potentially sustainable manufacturing route for alkaloid medicines and novel therapies. However, reaching commercially attractive titers, rates and yields remains challenging owing to the length and complexity of these metabolic pathways. One critical bottleneck is the low screening throughput and very high cost of the analytical methods used to quantify MIA for optimizing production. In this study, we evolved RamR, a promiscuous bacterial transcription factor to respond to five different MIAs, resulting in highly sensitive and selective sensor variants (EC50<10 M). X-ray crystallography and computational modeling provided insight into the MIA binding of the evolved biosensor variants. The RamR biosensing platform was functionalized in yeast and subsequently applied in a cost-effective semi-throughput screening campaign of a 188-gene overexpression library to identify high-performing cell factory designs for strictosidine, the common precursor for all MIAs. The fluorescent biosensor signal correlated with HPLC quantification (r2 = 0.932) allowing identification of single metabolic engineering hits which when combined yielded a maximum titer of >220 mg/L strictosidine, 3-fold higher than the parental reference strain. This study demonstrates the development of selective biosensors for MIAs and the cost-effective identification of novel metabolic engineering hits for optimizing MIA production in microbial cell factories.

BackgroundThe corn smut fungus Ustilago maydis is an important microbial model organism representing a genetically amenable and readily cultivable basidiomycete. Research in this fungus addresses a broad range of fundamental questions and its biotechnological exploitation is on the rise. Although genetic engineering in principle is well established, efficient methodology for synthetic biology approaches such as metabolic engineering or pathway transplantation has remained limited. ResultsHere, we present a comprehensive toolbox for U. maydis based on modular cloning and the characterization of more than 20 promoters. Careful comparative evaluation of insertion loci and terminator as well as reporter effects was conducted and a novel color-based strategy for straightforward genome integration was implemented. Moreover, the cloning and subsequent one-step integration of four transcriptional units into U. maydis was demonstrated by creating a "rainbow" strain producing four fluorescent proteins. ConclusionOverall, this next generation toolkit strongly advances genetic engineering and systems biology approaches in U. maydis, fostering its development into a valuable and competitive fungal chassis and prime model, particularly in applied research.

Muconic acid is a versatile platform chemical that can be biologically produced from lignocellulosic substrates, including from lignin-related aromatic compounds. Pseudomonas putida has been previously engineered to convert lignin-related aromatic compounds to muconate at quantitative molar yields. This high atom efficiency requires a supplemental carbon and energy source to support bacterial growth, and central carbon metabolic efficiency and its interaction with aromatic catabolism are underexplored. Here, we applied proteomics, metabolomics, and 13C-fluxomics to quantitatively compare central carbon and energy metabolism in wild-type P. putida KT2440 and a muconate-producing strain, P. putida CJ781. During cultivation on glucose and 4-hydroxybenzoate, CJ781 showed increased glucose uptake, reconfigured central fluxes, and increased extracellular leakage of aliphatic acids relative to wild type. These altered fluxes supported a 3-fold higher ATP pool, in excess of demand. Pyruvate and acetate secretion in CJ781 was mitigated by debottlenecking TCA-cycle entry via citrate synthase overexpression. Furthermore, tuned expression of the catechol dioxygenase and protocatechuate decarboxylase enabled the production of 36.3 g L-1 muconate at 1.1 g L-1 h-1. Overall, this work reveals how P. putida redirects carbon and energy fluxes to support aromatic bioconversion for improved bioproduction from renewable feedstocks.

{beta}-galactosidases (BGs) are essential enzymes widely used in the food industry, particularly in the production of lactose-free products. Among them, the BG from Aspergillus oryzae is of industrial relevance due to its activity at acidic pH and moderate thermal tolerance. However, enhancing its catalytic performance remains a key challenge. Traditional enzyme engineering methods are time-consuming and resource-intensive, limiting their scalability. Recent advances in Artificial Intelligence (AI), particularly those based on Natural Language Processing, offer a promising alternative by enabling efficient exploration of protein sequence space and prediction of beneficial mutations. In this study, we introduce an ensemble-based, zero-shot Protein Language Model pipeline that reconciles predictions from six independent models (ESM2 and the five ESM1v variants) combined with a diversity-aware candidate selection strategy. Applied to the BG from A. oryzae, this approach identified beneficial mutations leading to novel enzyme variants with up to a four-fold increase in catalytic efficiency on oNPGal, a two-fold increase on lactose, and, independently, a T338I variant with markedly enhanced thermostability ({approx}80% residual activity after 24 h at 60 {degrees}C), all without requiring supervised fine-tuning on experimental fitness data. Our results demonstrate that consensus across an ensemble of PLMs can efficiently enrich beneficial substitutions in industrially relevant enzymes and substantially reduce the number of wet-lab candidates that need to be screened. Table of Contents graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/726739v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@18084f7org.highwire.dtl.DTLVardef@99a102org.highwire.dtl.DTLVardef@19a64forg.highwire.dtl.DTLVardef@1f59cff_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cell-free protein synthesis (CFPS) is a powerful platform for synthetic biology, yet the factors governing reaction longevity remain poorly understood despite their importance for high-throughput applications. Here, the three principal determinants of CFPS performance--DNA template design, reaction composition, and lysate genotype--were systematically optimized to extend reaction lifetime in a 384-well plate format. Different energy regeneration systems were evaluated through real-time pH monitoring and metabolomic analyses to identify the metabolic constraints limiting prolonged protein synthesis. Lysates prepared from engineered Escherichia coli BL21(DE3) strains were further examined to assess the contributions of DNA, RNA, and amino acid stabilization. Systematic optimization of amino acid, nucleoside triphosphate, polyethylene glycol, and lysate concentrations identified DNA template stability and amino acid preservation as the primary factors sustaining CFPS activity. Combining these improvements yielded reactions that remained productive for >14 h and produced 567 {+/-} 64 g mL-1 active deGFP. These findings establish practical strategies for extending CFPS lifetime and improving high-throughput cell-free platforms.

The increasing accumulation of polyethylene terephthalate (PET) waste has prompted the development of sustainable biotechnological strategies for its degradation and valorisation. This study presents an integrated approach combining enzymatic PET depolymerization by Yarrowia lipolytica, engineered to express and secrete the cutinase HiC and the lipase CalB, with the microbial valorization of PET-derived monomers, terephthalic acid (TPA) and ethylene glycol (EG), by Rhodococcus jostii RHA1. Y. lipolytica was further engineered for xylose metabolism, enabling enzyme production from low-cost lignocellulose-derived substrates. Enzymatic assays with HiC and CalB crudes effectively hydrolysate PET to TPA and EG, demonstrating functional enzymatic activity without purification steps. In addition, R. jostii RHA1 was able to use as substrate the released monomers and accumulated intracellular lipids. Overall, this work demonstrates the feasibility of coupling the production of PET degrading enzymes and microbial lipid, using an abundant monosaccharide, with the assimilation of the PET degradation products to also produce microbial lipids. This modular system provides a promising framework for the sustainable upcycling of plastic waste into value-added bioproducts within a circular economy.

At neutral pH, Clostridial fermentative catabolism is typically acidogenic, with a product profile dominated by acetate and butyrate. H2 acts as a terminal electron acceptor via hydrogenases, which increases ATP-producing potential from glucose. Acetate production is characterised by both higher ATP and H2 yields, rendering it desirable but more thermodynamically limited. For this reason, is commonly understood that Clostridia can adjust the ratio of acetate to butyrate (Ace:But) produced to maximise ATP while maintaining sufficient pathway driving forces to sustain a high flux. We identify three redox-balanced product profiles that underlie the spectrum of Clostridial catabolic Ace:But ratios: Homoacetic (Ace:But = 2:0), Equimolar (0.67:0.67), and Homobutyric (0:1). To reach Ace:But ratios intermediate to these, the elementary flux modes (EFMs) underlying the aforementioned product profiles must be blended. We performed a maximum-minimum driving force (MDF) analysis to test the thermodynamic favourability of the pathways underlying different Ace:But ratios at varying H2 partial pressures (pH2). We find that blended EFMs are less efficient than their constituent EFMs at all pH2, allocating excessive driving force (DF) to certain reactions, thereby lowering the DF of others. This is, in part, due to the co-occurrence of hydrogenases with different optimal redox carrier ratios. One hydrogenase inevitably has very high DF, which decreases the DF available for other reactions. This leads to a lower minimum DF and a higher enzyme cost for operating blended EFMs. This implies that certain discrete Ace:But ratios are most favourable for large ranges of pH2, contradicting the continuity assumption in literature.

Saccharomyces cerevisiae is an excellent microbial platform for sustainable production of next generation biofuels such as the branched chain higher alcohols (BCHAs) isobutanol and isopentanol. A cytosolic pathway for BCHA production is generated from expression of prokaryotic orthologs of branched-chain amino acid (BCAA) enzymes acetolactate synthase (ALS), mutant NADH-dependent ketol-acid reductoisomerase (KARIP2D1-A1), and dihydroxy-acid dehydratase (DHAD). The potential for this pathway has been hindered by the availability of iron-sulfur clusters, particularly the 2Fe-2S cluster, required for DHAD to function in the cytosol. ILV3, the endogenous yeast DHAD located in the mitochondria, can be deleted to create a valine auxotroph. In this study we use bioinformatics, heterologous gene library synthesis, and a valine complementation assay to find prokaryotic iron-sulfur cluster biosynthetic gene clusters (BGC) and accessory genes that aid DHAD function in the yeast cytosol. This work presents, to our knowledge, the first functional BGC that enhances the cytosolic activity of prokaryotic DHADs in S. cerevisiae. The SUF BGC from Bacillus subtilis combined with a ferritin-like protein (FTNB) from Escherichia coli and the Lactococcus lactis DHAD enhanced the production of BCHAs. Combined expression gave an average isobutanol titer of 412mg/L, 1.8-fold greater than L. lactis DHAD expressed alone. This work establishes a blueprint for better biofuel production by improving iron-sulfur cluster dependent enzyme activity in the yeast cytosol.

Anaerocellum (formerly Caldicellulosiruptor) bescii, an anaerobic, extremely thermophilic (Topt [~]78 {degrees}C) lignocellulolytic bacterium, is a promising chassis for metabolic engineering and next-generation bioprocessing. Yet, a lack of well-characterized genetic parts in A. bescii has hampered metabolic engineering efforts. Here, using a previously developed hyperthermophilic {beta}-galactosidase reporter system, we screened a diverse panel of putative A. bescii promoter sequences, identifying promoters that drove reporter output across a broad range. For a select subset, we mapped their transcriptional start sites (TSSs) and evaluated ribosome binding site (RBS) regions using chimeric promoter constructs. By constructing truncated promoter variants, we defined functional regions within the widely used, high-expression S-layer protein promoter (Pslp) and engineered a compact 99 bp variant that retained substantial reporter activity. Finally, we demonstrated that these new promoters can be used for metabolic engineering by using two newly characterized promoters to express an established thermostable alcohol dehydrogenase from Thermoclostridium stercorarium to drive ethanol production in A. bescii. Together, this work expands and diversifies the A. bescii genetic toolkit, opening doors to future metabolic engineering efforts in this species.

The accumulation of plastic waste necessitates innovative strategies that convert polymer carbon into value-added products. Here, we present a sequential yeast-bacterial workflow for the integrated valorisation of glycerol and amorphous polyethylene terephthalate (amPET) into triacyl glyceride (TAGs) and polyhydroxyalkanoates (PHAs). First, an engineered obese strain of Yarrowia lipolytica was cultivated on glycerol, for the simultaneous production of intracellular lipids, up to 42.9% of its cell dry weight, and secretion of a PET-depolymerizing enzymatic cocktail, composed of the cutinase from Mycothermus thermophilus (HiC) and the lipase B from Moesziomyces antarcticus (CALB). The resulting enzymatic crude hydrolysed amPET, and the released degradation products--terephthalic acid (TPA) and ethylene glycol (EG)-- served as feedstocks for a bacterial consortium composed of Comamonas testosteroni RW31 and Pseudomonas putida JM37, which naturally assimilate TPA and EG, respectively. This consortium successfully upcycled the released monomers into intracellular polyhydroxybutyrate (PHB) and medium-chain-length PHAs. Furthermore, fluorescent strains of both bacteria enabled the development of a semi-quantitative method for monitoring the consortium population dynamics. Overall, this study provides a robust proof-of-concept for a circular bioeconomy approach, successfully coupling glycerol-based enzyme and lipid production with the downstream biological conversion of PET-derived monomers into valuable bioplastics. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/729029v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1575eedorg.highwire.dtl.DTLVardef@1195aeborg.highwire.dtl.DTLVardef@1a01bb0org.highwire.dtl.DTLVardef@79f4f6_HPS_FORMAT_FIGEXP M_FIG C_FIG

Engineering the ethanologenic Gram-negative bacterium Z. mobilis for the secretion of hydrolytic enzymes is a key step towards establishing a biofuel cell factory that uses complex waste material as feedstock. Secretion strategies in Z. mobilis have exclusively relied on signal peptides, which limit protein transport to the periplasm. To achieve single-step secretion across the Z. mobilis double-layered membrane, we sought to identify a native Type I Secretion System (T1SS) tag for fusion to proteins of interest. While a T1SS operon had been identified in the Z. mobilis genome, its native cargo had remained unknown and the use of T1SS secretion tags had so far been unexplored. Here, bioinformatic analysis identified the Major Intrinsic Protein (MIP) as a putative T1SS cargo, and its role validated through fusion of C-terminal sequences of two lengths (61 and 141 amino acids) to a heterologous {beta}-galactosidase from Bacteroides thetaiotaomicron, expressed in Z. mobilis. The 141 amino acid tag, including two RTX domains, resulted in significantly higher secretion efficiency than the 61 amino acid tag lacking RTX repeats, consistent with the established role of RTX domains in preventing premature cytoplasmic folding, thus improving secretion. As extracellular secretion of hydrolytic enzymes has remained a major bottleneck in the development of Z. mobilis as a sustainable cell factory, the identification of a native T1SS secretion tag directly addresses this limitation, introducing a novel tool for enzyme delivery.

Streptomyces bacteria produce a variety of secondary metabolites that hold clinical and agricultural value, yet their biosynthetic potential remains unrealized as many biosynthetic gene clusters are not expressed under standard laboratory conditions. Expression of these clusters is tightly regulated, often by cluster situated transcription factors. The TetR family are regulators whose activity is modulated by small molecule elicitors. Although many TetRs have been characterized, elicitors have only been identified for a small fraction of them. This lack of data presents a limitation in our ability to exploit elicitor-regulator pairs for activation of silent clusters and underscores the need for predictive and testable strategies for elicitor identification. In this work, we test the use of sequence similarity networks (SSNs) as a predictor of elicitor identity using the well characterized TetR protein, JadR2, that has a known elicitor, chloramphenicol. We utilized SSNs to identify JadR2 homologs that may also be elicited by chloramphenicol. We developed a heterologous Escherichia coli reporter system in which regulator activity was monitored using an EGFP readout of DNA binding activity. Using this system, we screened JadR2 and four homologs for responsiveness to chloramphenicol. We found that 3 homologs were elicited by chloramphenicol, all of which were formerly uncharacterized. These results demonstrate that TetR-family proteins can share elicitor responsiveness and that SSNs can be used to prioritize regulators for functional screening. This work establishes a genomics-informed and bioinformatics-guided framework for linking elicitors to their regulator, expanding the toolkit for natural product discovery by unlocking regulatory information across Streptomyces.

Acetate metabolism drives mixotrophic and heterotrophic growth in some microalgae. Acetyl-CoA synthetase (ACS) and acetate kinase (ACK) are often considered the main enzymes involved in acetate catabolism in microalgae; however, their contributions to metabolic flux and carbon allocation are not fully understood. In this study, the functions of cytosolic ACS1 and mitochondrial ACK2 were characterized using two knockout mutants of the model microalga Chlamydomonas reinhardtii. The acs1 mutant exhibited a growth-oriented phenotype, characterized by 29.8% faster cell growth at 96 h and up to a 15.5% higher acetate depletion rate, yet showed a 38.3% lower triacylglycerol (TAG) content at 48 h under heterotrophic conditions. By contrast, the ack2 mutant exhibited an altered carbon-allocation phenotype under heterotrophic conditions. Despite an up to 32.4% lower respiratory oxygen consumption rate and a 27.7% reduction in cell density, ack2 exhibited a 39.3% higher biomass concentration and a 90.4% greater dry weight per cell than the wild type at 96 h. Biochemical analysis revealed that ack2 accumulated 23.3% more carbohydrate than the wild type at 120 h under heterotrophic conditions, whereas its TAG level remained comparable to that of the wild type. These findings suggest that, under heterotrophic conditions, the loss of cytosolic ACS1 facilitates cell growth and division at the expense of TAG biosynthesis, whereas the loss of mitochondrial ACK2 regulates growth by affecting carbon flux toward biomass and carbohydrate accumulation. This work provides insight into acetate catabolism in C. reinhardtii and suggests targets for engineering microalgae for production of biomass and bioproducts.