Inducible flippase-mediated metabolic engineering of Rhodosporidium toruloides for enhanced 3-hydroxypropionic acid production from corn stover hydrolysate.

Advanced approaches for mitigating impact of pre-treatment generated inhibitors in lignocellulosic hydrolysates: A comprehensive review

Yeast-derived biomolecules are redefining the boundaries of green nanotechnology. Biosurfactants, exopolysaccharides, enzymes, pigments, proteins, and organic acids—when sourced from carbohydrate-rich lignocellulosic hydrolysates—offer a molecular toolbox capable of directing, stabilizing, and functionalizing nanoparticles (NPs) with unprecedented precision. Beyond their structural diversity and intrinsic biocompatibility, these biomolecules anchor a paradigm shift: the convergence of biorefineries with nanotechnology to deliver multifunctional materials for the circular bioeconomy. This review explores: (i) the expanding portfolio of metallic and metal oxide NPs synthesized through yeast biomolecules; (ii) molecular-level mechanisms of reduction, capping, and surface tailoring that dictate NP morphology, stability, and reactivity; (iii) synergistic roles in intensifying lignocellulosic processes—from enhanced hydrolysis to catalytic upgrading; and (iv) frontier applications spanning antimicrobial coatings, regenerative packaging, precision agriculture, and environmental remediation. We highlight structure–function relationships, where amphiphilicity, charge distribution, and redox activity govern resilience under saline, acidic, and thermally harsh industrial matrices. Yet, critical bottlenecks remain: inconsistent yields, limited comparative studies, downstream recovery hurdles, and the absence of comprehensive life-cycle and toxicological evaluations. To bridge this gap, we propose a translational roadmap coupling standardized characterization with real hydrolysate testing, molecular libraries linking biomolecule chemistry to NP performance, and integrated techno-economic and environmental assessments. By aligning yeast biotechnology with nanoscience, we argue that yeast-biomolecule-driven nanoplatforms are not merely sustainable alternatives but transformative solutions for next-generation lignocellulosic biorefineries.

Oleaginous filamentous fungi, such as Mucor circinelloides, are capable of accumulating high levels of single cell oil (SCO), making them attractive candidates for the production of biodiesel and other oleochemicals. Lignocellulosic feedstocks offer an abundant and cost-effective carbon source for SCO production due to their high polysaccharide content. However, most oleaginous microorganisms cannot directly utilize cellulose and hemicellulose polysaccharides, necessitating their conversion into monosaccharides. Lignocellulosic substrates can be saccharified either separately from fermentation (separate hydrolysis and fermentation; SHF) or simultaneously (simultaneous saccharification and fermentation; SSF). This study evaluated SSF using M. circinelloides, as well as SHF cultivations on two types of lignocellulosic hydrolysates, and two control fermentations, with process monitoring via four vibrational spectroscopy techniques: Fourier Transform Infrared (FTIR) spectrometer with fibre optic probe, FTIR microspectrometer, FTIR spectrometer with high throughput setting (HTS), and FT-Raman spectrometer with HTS. Quantitative estimation of glucose in the cultivation media and lipid content in the biomass was achieved using PLSR analysis of FT-Raman measurements from the cell suspension. FT-Raman spectroscopy demonstrated exceptional capability for online and at-line process monitoring among the tested techniques. It enabled direct and rapid analysis of raw cell suspensions (containing growth media, cellulose-rich pulp substrate, and fungal biomass) without the need for sample pre-treatment, purification, or modification. FT-Raman provided comprehensive biochemical profiles, effectively detecting key chemical changes in both the cellulose-rich pulp substrates and the fungal biomass, including lipid accumulation by the oleaginous fungi. FTIR with fiber optics is effective for monitoring glucose in SHF processes, but its accuracy is limited in SSF processes due to the very low glucose concentrations. The study demonstrates that FTIR microspectroscopy is a valuable tool for lab-scale fermentation process development, as well as for investigating the bioconversion of lignocellulosic biomass into fungal biomass and metabolites. FT-Raman spectroscopy is highlighted as a powerful process analytical technology (PAT) tool for real-time or near-real-time monitoring of SSF processes for intracellular SCO production. Its ability to provide rich chemical information rapidly and without extensive sample preparation holds significant promise for optimizing industrial SCO production from lignocellulosic feedstocks.

Fine-tuning of gene expression is often required to achieve competitive production levels in microbial cell factories. Several orthogonal expression systems based on heterologous regulatory parts have been developed for Saccharomyces cerevisiae. In laboratory conditions the systems demonstrate predictable results, but few expression systems have been tested in industrial conditions. Here, a new expression system based on the bacterial gene cusR was developed for S. cerevisiae, and two previous developed systems, the strong Bm3R1-based system and the quinic acid inducible Q-system, were adapted for compatibility with the Yeast MoClo Toolkit. The bacterial transcription factors CusR and Bm3R1 acted as DNA binding domains, and fused to a viral activation domain, they functioned as transcriptional activators. The Q-system is originally from Neurospora crassa and consists of a transcriptional repressor, QS, which in the absence of quinic acid blocks the activity of a transcriptional activator, QF2. Quinic acid binds to QS, inhibiting QS from blocking the activity of QF2 in a dose-dependent manner. The gene expression systems were assessed in industrially relevant conditions, proving a predictable performance at low pH. The performance of the constitutive systems was predictable also at high temperature and in a synthetic lignocellulosic hydrolysate medium. Altogether, the MoClo-compatible expression systems enable fast construction of fine-tuned production pathways for S. cerevisiae cell factories used for industrial applications.

New insights into the synergistic effect of Mg2+ and sulfonated lignin on the enzymatic hydrolysis of lignocellulose.

Electro-fermentation driven detoxification of furfural to methane.

Enhanced lignocellulose hydrolysis and controlled biogas-to-platform chemical switching through temperature-driven microbial specialization in hyperthermophilic anaerobic digestion

Isolation, cloning, and characterization of a novel GH5 cellulase from yak rumen metagenome for enhanced lignocellulose hydrolysis in biofuel production and ruminant feed utilization.

Efficient production of biobased monomers from lignocellulosic hydrolysates is considered an important approach to reducing product costs and achieving sustainable biomanufacturing. However, the low conversion efficiency leads to a low yield and extended fermentation cycles. Here, we enhanced the utilization efficiency of xylose by strengthening the isomerase pathway. Subsequently, we successfully developed an efficient strain capable of synchronous glucose-xylose utilization for cadaverine production, thereby reducing the fermentation time from 30 to 24 h. Through optimization of the fermentation process, cadaverine production reached the highest level of 60.74 g/L in glucose-xylose mixture fermentation, with a yield of 0.34 g/gsugar and a total sugar utilization rate of 3.65 g/L/h in a 5 L fermenter. When applied to the fermentation of corn stalk hydrolysate, 24.42 g/L cadaverine was obtained, with a yield of 0.32 g/gsugar. This work has laid the foundation for the large-scale production of biobased monomers from lignocellulosic hydrolysates.

One of the major uses of petroleum products is transportation fuel such as gasoline and diesel, consumption of which substantially contributes to global warming. Oleaginous microorganisms capable of growing on lignocellulose hydrolysates serve as sustainable alternatives to hydrocarbon derived fuels. Co-culturing of two oleaginous microorganisms Acinetobacter baylyi ADP1 and Lipomyces starkeyi Y-1389 potentially allow us to overcome limitations of each of them, i.e., a relatively low lipid productivity for the former and a sensitivity to lignin hydrolysate products of the latter. This study investigated the ability of L. starkeyi Y-1389 to produce lipids alone or in co-culture with A. baylyi ADP1, specifically examining the influence of lignin-derived inhibitors on this process. Microorganisms were cultured in a mineral medium supplemented with sugar mixtures (glucose and xylose) and non-sugar components (acetate, formate, furfural, 5-hydrox ymethylfurfural, p-hydroxybenzaldehyde, syringaldehyde and vanillin), which are typically produced during lignocellulose hydrolysis. Both microbes were shown to tolerate some levels of furfural (up to 0.5g/l for yeasts and 0.1g/l for bacteria), 5-hydroxymethylfurfural (up to 0.5g/L for both), p-hydroxybenzaldehyde (up to 0.25g/1 for bacteria and 0.5g/ for yeasts), syringaldehyde (up to 0.5g/l for both) and vanillin (up to 0.1g/l for yeasts and 0.5g/l for bacteria), which typically serve as potential growth inhibitors. Formate and acetate, also common components of lignocellulose hydrolysates, was shown to suppress lipid production in A. baylyi ADP1 but not L. starkeyi Y-1389. During 144h fermentation all potential inhibitors as well as acetate and formate were completely consumed by co-cultures. While lipid content in co-culture (32-36% of dry biomass) was comparable to L. starkeyi Y-1389 monoculture (32-40%), without significant advantages observed for co-culture in terms of yield, the co-culture exhibited higher resistance to a lignin-derived inhibitor mix compared to the yeast monoculture. This suggests that A. baylyi ADP1 effectively detoxifies the medium, allowing for better overall performance under inhibitory conditions. The results also suggest that wood hydrolysates are more favorable for lipid production using the A. baylyi ADP1 and L. starkeyi Y-1389 co-culture than hydrolysates of herbaceous plants indicating that a co-culture is a better choice for biotechnological production of lipids when wood hydrolysate is used. Our findings indicate that despite bacterial dominance and suppression of A. baylyi lipid production by acetate and formate, the co-culture approach is a promising strategy for biotechnological lipid production, particularly when utilizing wood hydrolysates, warranting further investigation into optimizing these microbial consortia.

The coutilization of multiple carbon sources improves metabolic flexibility and efficiency, thereby enhancing product yields while alleviating cellular stress and energy imbalances during biosynthesis. In this study, we engineered Yarrowia lipolytica for enhanced β-carotene production by coutilizing xylose and acetic acid─two major carbon components derived from lignocellulosic hydrolysates. Through the integration of heterologous xylose assimilation pathways, a β-carotene biosynthetic module, the native Acs/Aarc-mediated acetyl-CoA synthesis route, and the nonoxidative glycolysis (NOG) pathway, we established a robust metabolic framework to optimize carbon flux. The engineered strain produced 185.4 mg/L (27.8 mg/g dry cell weight) β-carotene from xylose alone, which was enhanced to 4.2-fold to 776.9 mg/L with a content of 70.4 mg/g dry cell weight under cofermentation with 25 g/L xylose and 25 g/L sodium acetate. These results demonstrate the potential of Y. lipolytica as a versatile microbial chassis for the bioconversion of renewable carbon sources into high-value products, and offer a promising strategy for lignocellulosic biorefinery development through C5-C2 coutilization coupled with NOG pathway enhancement.

Lactic acid is an important biobased chemical widely used in the production of biodegradable plastics, food, and pharmaceuticals. However, the application of flocculant Saccharomyces cerevisiae remains limited in addressing stresses such as high-glucose and inhibitor-rich conditions derived from biomass, particularly in D-lactic acid (D-LA) production. This study investigates two genetically engineered S. cerevisiae F118 strains, ΔCYB2::LpDLDH and ΔPDC1::LpDLDH, for D-LA production under high-glucose and inhibitor-stress conditions that mimic lignocellulosic hydrolysates in shake-flask fermentation. At 150 g/L glucose, ΔCYB2::LpDLDH produced 41 ± 0.73 g/L D-LA, whereas ΔPDC1::LpDLDH yielded 80 ± 1.78 g/L, corresponding to 27% and 53% of the theoretical yield, respectively. Calcium carbonate (CaCO3) supplementation enhanced glucose consumption and strengthened flocculation in ΔPDC1::LpDLDH. The addition of 5% inhibitory chemical compounds (ICCs) consisting of furfural, HMF, and weak acids redirected carbon flux in ΔCYB2::LpDLDH toward D-LA formation and reduced ethanol byproduct accumulation. Transcriptomic analysis revealed the upregulation of stress-response genes (HOG1, TPS1) and cell-wall remodeling genes (CRH1, SCW10) in response to high-glucose stress. The strongly flocculent F118ΔCYB2::LpDLDH strain exhibited greater tolerance to weak acids and furfural than the weakly flocculent F118ΔPDC1::LpDLDH strain. Metabolomic profiling indicated that under inhibitor stress, carbon flux was diverted from the TCA cycle toward lactate synthesis to maintain redox balance. These findings highlight the multifaceted benefits of flocculation in enhancing strain robustness and D-LA productivity under harsh fermentation environments, providing insights for developing resilient yeast platforms for lignocellulosic bioprocessing.

Improving the reductive tricarboxylic acid (rTCA) pathway in facultative anaerobic bacteria has important implications for efficient succinic acid (SA) production. With respective to enhance SA production from lignocellulosic hydrolysates via improving the rTCA pathway of Klebsiella oxytoca, the overexpression of phosphoenolpyruvate carboxykinase (PCK) and carbonic anhydrase (CA) genes coupling with supplementation of sodium bicarbonate (NaHCO3) were performed. The optimal concentrations of NaHCO3 supplementation for wild (WT) and engineered (RT-pck, RT-ca.) strains were determined to be 3g/L, 1g/L and 4g/L, respectively. The SA production achieved by RT-pck and RT-ca. strains with respective optimal NaHCO3 supplementation reached 28.00 ± 0.33g/L and 29.82 ± 0.35g/L at 72h of fermentation, resulting in the peak PCK enzyme and CA enzyme activities of 76.52 ± 6.36 pmol/(min·104 cells) and 47.06 ± 8.99 pmol/(min·104 cells), separately, as well as upregulation of several genes associated with the rTCA pathway. These findings elucidate the synergistic mechanism of pck/ca. gene overexpression and NaHCO3 supplementation in improving the rTCA pathway to enhance SA production. Overall, this study provides an effective strategy for improving lignocellulosic hydrolysate-based SA production, offering promising applications in lignocellulosic biorefinery and bioproduct process.

Development of iterative genome-editing method based on a Rock-Paper-Scissors strategy in Clostridium tyrobutyricum for butyl butyrate from lignocellulosic biomass hydrolysate

Phenolic compounds from lignin degradation in harsh pretreatment of lignocellulose persistently exist at low concentrations in hydrolysates even after standard detoxification and enzymatic hydrolysis. This study found that minor phenolic compounds in dry acid-pretreated and biodetoxified wheat straw hydrolysates were the key inhibitors of Escherichia coli (E. coli). Multiple E. coli strains exhibited poor cell growth and metabolic activity in wheat straw hydrolysates with the presence of minor phenolics. However, the cell growth and metabolic activity of E. coli strains were significantly recovered with the declining phenolic content. Several recombinant E. coli strains successfully fermented 22.17g/L of ethanol and synthesized 12.04g/L of cadaverine using wheat straw hydrolysates after the removal of minor phenolics. These findings highlight the critical role of phenolic compounds in the inhibition of E. coli strains and provide a foundation for E. coli recombinants in biorefinery fermentations for biofuel and biochemical productions.

Acetic acid (AA) is a natural by-product of ethanol fermentation and widely exists in lignocellulosic hydrolysate as a fermentation inhibitor since its high concentration damages proteins and nucleic acids. The study of the action mechanisms of AA at a pH lower than its pKa (4.76) is particularly relevant since it disrupts the function of cellular membranes by altering the conformations of membrane proteins and lipid organization. This study aims to investigate the molecular mechanism of AA influence in industrial strains of S. cerevisiae (ATCC 9804 and ATCC 13007) depending on metabolic condition (fermentation versus respiration) and external pH (3․0 or 4.5). The results show that 10-50 mM AA reduces the viability of both strains studied. Moreover, the ATCC 13007 strain is more sensitive to AA stress compared to the ATCC 9804 strain. Yeast resistance to AA stress is higher under respiratory metabolism compared to fermentation and at higher pH. Catalase activity was observed to increase by 1.5-6-fold under AA stress conditions, which correlates with yeast growth changes. The influence of AA stress is reactive oxygen species-dependent, and redox balance regulation was found to increase yeast robustness to AA by 2-fold. The study anticipates valuable insights into yeast adaptation to stress conditions, contributing to the development of robust yeast strain construction for biotechnological advancements in bioethanol or yeast protein production.

Optimization of bacterial nanocellulose synthesis using surfactants for immobilization of Saccharomyces cerevisiae to enhance resistance to lignocellulose-derived inhibitor.

Analysis of glucose inhibition characteristics during high-solids enzymatic hydrolysis of pretreated lignocellulose

Zymomonas mobilis is a promising biocatalyst for the sustainable conversion of lignocellulosic sugars into biofuels and bioproducts, yet its response to lignocellulosic hydrolysates remains poorly understood. Here, we investigate the physiological response of Z. mobilis to ammonia fiber expansion (AFEX)-pretreated switchgrass hydrolysate using a systems-level approach integrating LC-MS/MS-based lipidomics and shotgun proteomics. Growth on hydrolysate induced substantial shifts in fatty acid and membrane phospholipid composition, alongside broad proteomic remodeling. Notably, Z. mobilis exhibited a stress response characterized by the upregulation of heat shock proteins and efflux transporters and the downregulation of cell motility proteins. Unexpectedly, hydrolysate exposure also led to a robust upregulation of the Entner-Doudoroff pathway, the ethanol fermentation pathway, and other central carbon metabolism enzymes, indicating a substantial cellular investment potentially driven by additional nutrient availability in hydrolysate. These findings provide new insights into the metabolic adaptations of Z. mobilis to lignocellulosic hydrolysates, informing strategies to enhance its biofuel production capabilities.IMPORTANCEBiomass pretreatment processes release fermentable sugars from lignocellulosic biomass, but they also generate inhibitors that can impact microbial metabolism. This study provides a systems-level evaluation of how Zymomonas mobilis responds to hydrolysate stress, revealing distinct physiological and lipid membrane remodeling responses. While some stress responses overlap with those induced by ethanol and isobutanol toxicity, both valuable biofuels, hydrolysate exposure elicits unique metabolic shifts. These findings offer valuable insights for engineering Z. mobilis strains with improved tolerance and performance for efficient bioconversion of lignocellulosic hydrolysates into biofuels and bioproducts.