Biotechnology for Biofuels

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.

Lignocellulosic biomass represents a promising renewable feedstock for sustainable biorefinery applications, yet efficient enzymatic saccharification remains challenging due to the recalcitrant structure of plant cell walls. This study presents a comprehensive comparative analysis of enzymatic activities, saccharification performance, and secretome composition of six fungal species cultivated under solid-state fermentation (SSF) and submerged fermentation (SmF) conditions using untreated flax shives as substrate. While SmF yielded approximately 4-fold higher total protein concentrations (0.38 {+/-} 0.13 g.L-1 vs. 0.08 {+/-} 0.02 g.L-1), SSF-derived enzymes demonstrated superior specific enzymatic activities, particularly for endo-xylanase and endo-cellulase, resulting in more efficient biomass saccharification. Proteomics analysis revealed distinct secretome profiles between fermentation modes, with SSF showing higher proportions of polysaccharide metabolism proteins (71.0%) compared to SmF (49.3%), while SmF exhibited greater enzyme diversity including more lytic polysaccharide monooxygenases (LPMOs) and auxiliary activity enzymes. Trichoderma species consistently demonstrated the highest saccharification efficiency, with glucose yields reaching 2.37 mM under SSF conditions. A Scheffe simplex-lattice mixture design comprising 65 enzyme cocktail combinations revealed significant synergistic interactions between several cocktails, with the binary mixture of Trichoderma 2SA21 and P. chrysogenum achieving 54% synergy - in terms of higher sugar release above expectations - and the highest total monosaccharide release (1.80 mM). These findings provide practical guidance for developing cost-effective enzyme cocktails for lignocellulosic biorefinery applications, emphasizing the importance of fermentation mode selection and strategic strain combination over enzyme supplementation complexity. The methodology established here, combining systematic screening, comparative proteomics, and statistical mixture design, offers a robust framework for optimizing fungal enzyme systems across diverse biomass substrates. BULLET POINTSSuperior enzymatic activity (xylanase, cellulase) and saccharification in solid-state fermentation Superior total protein content and diversity in submerged fermentation Specific enzyme cocktails combination can lead to synergistic effects, justifying a combinatorial approach GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/729743v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1f3704dorg.highwire.dtl.DTLVardef@151e4bforg.highwire.dtl.DTLVardef@180ced7org.highwire.dtl.DTLVardef@18bb42f_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

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.

The bacterium Zymomonas mobilis is widely studied for its potential as an industrial biofuel producer. Anoxic fermentation by Z. mobilis in lignocellulosic hydrolysate can generate bioethanol from renewable plant biomass. In this study, we deleted a gene from the Z. mobilis genome encoding a homolog of OxyR, a transcription factor that activates an oxidative stress response in bacteria to reduce reactive oxygen species (ROS). Deletion of this transcription factor inhibited growth of Z. mobilis in oxic, but not anoxic, conditions in laboratory media. A ROS probe revealed that the oxyR response is required to reduce intracellular ROS during oxic growth. Importantly for biofuel production, the absence of oxyR inhibited growth and delayed ethanol production during anoxic hydrolysate fermentation. To determine the source of oxidative stress in hydrolysate, we grew{Delta} oxyR in a synthetic hydrolysate containing known inhibitors found in hydrolysate. There was no growth defect in{Delta} oxyR in the synthetic hydrolysate, indicating that known inhibitory compounds are not the source of anoxic oxidative stress. We determined that Ammonia-Fiber Expansion (AFEX) switchgrass hydrolysate contains significant peroxide concentrations. Addition of catalase to hydrolysate improves growth of both{Delta} oxyR and wild-type Z. mobilis in hydrolysate. This study uncovers an important source of stress to Z. mobilis during biofuel fermentation. ImportanceFermentation of non-food biomass is a promising avenue for sustainable production of fuels and chemicals, but several challenges currently limit applicability of this technology. One major hurdle is that when biomass is deconstructed into a fermentable form, many byproducts are generated that inhibit microbial fermentation. Here, we investigated how a fermentative bacterium, Zymomonas mobilis, experiences oxidative stress during anoxic biomass fermentation, and identified genes important in this response. These findings provide a better understanding of the stresses faced by Z. mobilis during biofuel production. Fully understanding the effects of hydrolysate on biofuel-producing microbes is crucial for optimizing production and making carbon-neutral fuel a reality.

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.

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.

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.

Engineering the biosynthesis of fully biodegradable polyhydroxyalkanoates (PHAs) from non-food and renewable feedstocks like lignocellulose is becoming an attractive strategy for sustainable biomanufacturing. However, the efficiency and diversity of PHA synthesis from lignocellulosic hydrolysate (LH), mainly containing glucose and xylose, still remains challenge. Here, Halomonas TD, a cost-effective PHA-producing chassis, was developed to utilize glucose and xylose (or LH) for effective production of poly-3-hydroxybutyrate (PHB) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] by engineering the phosphoketolase pathway-dependent xylose metabolism in the genome, yielding 50.1 g L-1 PHB and 42.6 g L-1 P(3HB-co- 11.4 mol% 4HB) under fed-batch condition. Subsequently, the introduction of Weimberg pathway was found be able to synthesize terpolymer consisting of 3-hydroxybutyrate (3HB), 4-hydroxybutyrate (4HB) and 3-hydroxyvalerate (3HV), namely [P(3HB-co-4HB-co-3HV)], from glucose and xylose only due to the isoenzyme activity of keto-acid decarboxylase encoded by kivD, which converts xylose-derived intermediate 2,5-dioxopentanoate and -ketoglutarate into butanedial (4HB synthesis) and 2-ketobutyrate (3HV synthesis), respectively. Finally, a tailored-made xylose-induced system was constructed to achieve exquisite xylose transmembrane transportation control for improved synthesis of terpolymer P(3HB-co-4.5 mol% 4HB-co- 3.0 mol% 3HV), reaching to 6.3 g L-1 under shake-flask condition, together with the co-expression of fine-tuned phosphoketolase and Weimberg pathways. This study provides a feasible and sustainable alternative for lignocellulosic resources valorization powered by the engineered Halomonas TD capable of efficient xylose utilization and diverse PHAs synthesis.

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

Fermentation of C1 gases is an emerging technology where waste gases are bio converted into value-added products. This study navigates the gas fermentation potential of Gordonia rubripertincta to produce carotenoids. The crucial carbon monoxide dehydrogenase (CODH) enzyme, necessary for gas uptake by the microbe, was found to be present in G. rubripertincta through blastp on NCBI website. The organism was then used for gas fermentation experiments in a continuous stirred tank reactor (CSTR) in different modes of reactor operation resulting in the production of about 500 mg pigment/g WCW (wet cell weight). Two important reactor parameters, molybdenum content and pH, were optimized for enhanced carotenoid production. Overall, G. rubripertincta was observed to be an efficient candidate organism for C1 gas fermentation. KEY HIGHLIGHTSO_LIGordonia rubripertincta synthesises aerobic carbon monoxide dehydrogenase enzyme. C_LIO_LIIt is a potential gas fermenting microbe that gives carotenoids as product. C_LIO_LIThe gas uptake efficiency of the microbe is more in fed-batch discontinued mode. C_LIO_LIIn FB-D, the resultant carotenoids are 500+9 mg/g wet cell weight (WCW). C_LIO_LIMo/pH of 20 mg/7.0 resulted in highest carotenoids, i.e., 134+41 mg/g WCW. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/722808v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@8b1185org.highwire.dtl.DTLVardef@2b6f90org.highwire.dtl.DTLVardef@1a9697dorg.highwire.dtl.DTLVardef@14c9dc8_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

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.

Mixed-linkage {beta}-glucans (MLGs) are emerging as promising biopolymers with significant biotechnological potential due to their unique structural and rheological properties. In rhizobia, MLG biosynthesis is controlled by the second messenger cyclic di-GMP (c-di-GMP) and mediated by the bicistronic operon bgsBA. However, the full composition of the biosynthetic machinery and strategies for enhanced production remain incompletely understood. In this study, we demonstrate that the outer membrane protein TolC is essential for MLG production in Sinorhizobium meliloti. Genetic disruption of tolC abolished MLG synthesis, while its complementation restored production. We propose that TolC forms a tripartite complex with BgsA and BgsB, enabling efficient polymer synthesis and export. Furthermore, co-overexpression of tolC, bgsBA, and a constitutively active diguanylate cyclase (pleD*) yielded a 10-fold increase of MLG over a control plasmid without tolC, reaching up to [~]10 g/L under bioreactor conditions. Additionally, this genetic module enabled de novo MLG production in otherwise non-producer rhizobial hosts (e.g. Mesorhizobium japonicum), allowing bacterial chassis exchanges and highlighting its portability and potential for synthetic biology applications. Overall, our findings identify TolC as a key component of the MLG biosynthetic machinery and provide a robust platform for the scalable production of this valuable biopolymer. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/721817v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@1da8e1org.highwire.dtl.DTLVardef@13a7b06org.highwire.dtl.DTLVardef@62d6eeorg.highwire.dtl.DTLVardef@10cc02d_HPS_FORMAT_FIGEXP M_FIG C_FIG

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

Malic acid is a C4 dicarboxylic acid traditionally produced from petroleum and widely used in the food industry. As a sustainable alternative, it can also be produced as a value-added platform chemical from biomass. Previously, the Escherichia coli strain XZ658 was engineered to produce L-malate via the carbon-fixation reductive branch of the TCA cycle. In this study, we further improved this system by relieving allosteric regulation of citrate synthase, addressing redox imbalance, and enhancing malate export. These modifications approximately doubled the L-malate titer in the final strain MO128 compared to XZ658 under simple batch fermentation conditions. The process achieved a high mass yield of 1.2 g malate g-{superscript 1} glucose, highlighting the carbon-fixation capacity of the reductive TCA pathway for fermentative malate production.

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.

Extremophilic red microalgae are promising platforms for sustainable biotechnology, combining robust growth under selective thermoacidophilic conditions with production of thermostable phycobiliproteins and carbon-rich biomass. However, reactor-dependent effects on growth, product formation and biomass composition remain insufficiently resolved. Here, we systematically evaluated the extremophilic red microalga Cyanidioschyzon merolae across cultivation scales and reactor formats and benchmarked its performance against the well-established Galdieria javensis and Limnospira platensis. In small-scale multi-cultivator photobioreactors and microfluidic growth chambers, C. merolae showed superior growth, reaching a maximum growth rate of 0.034 {+/-} 0.001 h-1 and 8.3 {+/-} 0.3 g l-1 cell dry weight. Microfluidic cultivation enabled growth analysis at single-cell resolution and matched growth rates obtained in photobioreactors. To identify scalable production strategies, C. merolae was further cultivated in a flat-panel photobioreactor and a custom-designed internally illuminated photobioreactor. The custom-designed photobioreactor delivered the highest biomass concentration and productivity, yielding 11.5 {+/-} 0.6 g l-1 cell dry weight and 1.07 {+/-} 0.06 g l-1 d-1, and comparable yields with regard to R-phycocyanin and R-allophycocyanin. Biomass analysis revealed substantial carbon and nitrogen contents, starch accumulation up to > 20 % of cell dry weight, and fatty acids dominated by palmitic, linoleic and oleic acids. Despite its reduced cell wall fraction, C. merolae contained structurally diverse, cultivation-dependent polysaccharides. These results establish C. merolae as a versatile chassis for thermostable pigment production and renewable feedstock generation, highlighting photobioreactor design as a key determinant of productivity and biomass quality.

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.