There is an urgent need to develop bioprocesses independent of fossil resources to address resource depletion and mitigate environmental harm. Transitioning to a bio-based economy requires prioritizing chemical production processes that utilize renewable resources, ensuring sustainability and environmental responsibility. 5-Hydroxymethylfurfural (HMF) and its derivatives are promising building blocks, ranked among the top 12 bio-based molecules derived from biomass. This study investigates the potential of wine residues as substrates for HMF production and explores the yeast Saccharomyces cerevisiae, a robust industrial microbial cell factory, as a whole-cell biocatalyst for converting HMF into high-value compounds, offering an alternative to chemical synthesis.
Several S. cerevisiae strains were compared for their ability to convert HMF, demonstrating varying capacities for oxidation or reduction. For the first time, HMF derivatives with potential industrial applications were produced using an HMF-rich hydrolysate obtained from sustainable processing of wine-growing waste, such as grape pomace and must surplus. The selected yeast strain was engineered to express the oxidoreductase enzyme of HMF/Furfural from Cupriavidua basilensis strain HMF14, resulting in a 15-fold increase in the accumulation of oxidized derivatives such as 2,5-furandicarboxylic acid (FDCA).
These findings highlight the potential of leveraging wine residues and engineered S. cerevisiae strains to develop sustainable bioprocesses for producing valuable HMF derivatives, thereby contributing to the advancement of bio-based chemical production.
Feruloyl esterases (FEs, EC 3.1.1.73) play a crucial role in biological synthesis and metabolism. However, the identification of versatile FEs, capable of catalyzing a wide range of substrates, remains a challenge. In this study, we obtained 2085 FE sequences from the BRENDA database and initiated with an enzyme similarity network analysis, revealing three main clusters (1–3). Notably, both cluster 1 and cluster 3 included the characterized FEs, which exhibited significant differences in sequence length. Subsequent phylogenetic analysis of these clusters unveiled a correlation between phylogenetic classification and substrate promiscuity, and enzymes with broad substrate scope tended to locate within specific branches of the phylogenetic tree. Further, molecular dynamics simulations and dynamical cross-correlation matrix analysis were employed to explore structural dynamics differences between promiscuous and substrate-specific FEs. Finally, to expand the repertoire of versatile FEs, we employed deep learning models to predict potentially promiscuous enzymes and identified 38 and 75 potential versatile FEs from cluster 1 and cluster 3 with a probability score exceeding 90%. Our findings underscore the utility of integrating phylogenetic and structural features with deep learning approaches for mining versatile FEs, shedding light on unexplored enzymatic diversity and expanding the repertoire of biocatalysts for synthetic applications.
Aspergillus oryzae is a widely used host for heterologous expression of fungal natural products. However, the vectors previously developed are not convenient for use and screening positive transformants by PCR and fermentation is time- and effort-consuming. Hence, three plug-and-play vectors were developed here for multi-gene expression and liquid chromatography mass spectrometry detection was introduced to screen positive transformants. Using rug BGC for verification, we demonstrated that the vectors we developed perform well and liquid chromatography mass spectrometry detection is feasible to screen positive transformants. For deleterious gene expression, PxyrA rather than PamyB was employed. Utilizing the toolkit described here to express natural products, dozen days can be saved.
The global trend toward carbon neutrality and sustainability calls for collaborative efforts in both the basic and applied research sectors to utilize mushroom mycelia as environmentally friendly and sustainable materials. Fungi, along with animals and plants, are one of the major eukaryotic life forms. They have long been utilized in traditional biotechnology sectors, such as food fermentation, antibiotic production, and industrial enzyme production. Some fungi have also been consumed as major food crops, such as the fruiting bodies of various mushrooms. Recently, new trends have emerged, shifting from traditional applications towards the innovative use of mushroom mycelium as eco-friendly bioresources. This approach has gained attention in the development of alternative meats, mycofabrication of biocomposites, and production of mycelial leather and fabrics. These applications aim to replace animal husbandry and recycle agricultural waste for use in construction and electrical materials. This paper reviews current research trends on industrial applications of mushroom mycelia, covering strain improvements and molecular breeding as well as mycelial products and the production processes. Key findings, practical considerations, and valorization are also discussed.
Amino acids are important bio-based products with a multi-billion-dollar market. The development of efficient high-throughput screening technologies utilizing biosensors is essential for the rapid identification of high-performance amino acid producers. However, there remains a pressing need for biosensors that specifically target certain critical amino acids, such as L-threonine and L-proline. In this study, a novel transcriptional regulator-based biosensor for L-threonine and L-proline was successfully developed, inspired by our new finding that SerE can export L-proline in addition to the previously known L-threonine and L-serine. Through directed evolution of SerR (the corresponding transcriptional regulator of SerE), the mutant SerRF104I which can recognize both L-threonine and L-proline as effectors and effectively distinguish strains with varying production levels was identified. Subsequently, the SerRF104I-based biosensor was employed for high-throughput screening of the superior enzyme mutants of L-homoserine dehydrogenase and γ-glutamyl kinase, which are critical enzymes in the biosynthesis of L-threonine and L-proline, respectively. A total of 25 and 13 novel mutants that increased the titers of L-threonine and L-proline by over 10% were successfully identified. Notably, six of the newly identified mutants exhibited similarities to the most effective mutants reported to date, indicating the promising application potential of the SerRF104I-based biosensor. This study illustrates an effective strategy for the development of transcriptional regulator-based biosensors for amino acids and other chemical compounds.
Kaempferol and quercetin possess various biological activities, making them valuable in food and medicine. However, their production via traditional methods is often inefficient. This study aims to address this gap by engineering the yeast Yarrowia lipolytica to achieve high yields of these flavonoids. We designed a kaempferol biosynthetic pathway by integrating multiple-copy fusion enzyme expression modules, F3H-(GGGGS)2-FLS, into the genome with an optimized linker (GGGGS)2 to enhance kaempferol production from naringenin. To synthesize quercetin de novo, we introduced the FMOCPR gene into the kaempferol-synthesizing strain using the optimized pFBAin promoter. Notably, increasing glucose concentration effectively boosted the production of both flavonoids. Our results demonstrated kaempferol and quercetin titers reaching 194.30 ± 7.69 and 278.92 ± 11.58 mg/L, respectively, in shake-flask cultures. These findings suggest that Y. lipolytica is a promising platform for the efficient production of flavonoid-derived products.
This research investigated the acidogenic fermentation (AF) of sugar cane molasses in an up-flow anaerobic sludge blanket (UASB) reactor for the production of carboxylates. The first step was to assess the optimum process temperature (25, 35 or 55 ºC) using two different granular inocula, one from a brewery company (BGS) and other from a paper plant company (PGS). These experiments determined that the most suitable temperature for carboxylates production was 25 ºC, obtaining higher bioconversions (27.3 ± 0.3% using PGS and 39.2 ± 0.2% using BGS), despite the low pH value recorded (4.0-4.2). Then, both inocula were tested in UASB reactors. As a consequence of the operational conditions (25 ºC, pH = 5.5-6, organic loading rate (OLR) = 3 gCOD·L-1·d-1 and hydraulic retention time (HRT) = 10 d), the microbial communities changed from those typical for biogas production to those specialised in the production of volatile fatty acids (VFAs). Indeed, the highest bioconversion efficiency (70.1%) was obtained with BGS, where uncultured Eubacteriaceae family microorganisms (56.0%) prevailed, enhancing the production of butyric acid (59.5 ± 2.4%w/w). Consequently, this inoculum was used to further identify the OLR threshold that should not be exceeded to attain optimal carboxylates production. OLR of 6 gCOD·L-1·d-1 resulted in a decrease in bioconversion efficiency (59.5%). The VFAs pool was dominated by butyric acid (63.0 ± 1.4%w/w at an OLR of 4.5 gCOD·L-1·d-1 and 52.8 ± 2.2%w/w at 6 gCOD·L-1·d-1). The microbial community became even more specialised, increasing the presence of Firmicutes and Actinobacteriota phyla, proving that the imposed conditions favoured the production of VFAs when operating semicontinuously fed UASB reactors.