Agricultural and industrial byproducts are abundant in bioactive compounds and can serve as alternative resources in producing a broad spectrum of value-added commodities, including biofuel, biogas, mushrooms, and tempeh, as demonstrated in numerous studies and industries. By utilising such waste by-products as stock materials, it is possible to reduce production costs and mitigate environmental pollution. These wastes are utilised in the creation of bioenergy fuels, vitamins, antibiotics, biocatalysts, antioxidants, and diverse commercially important types of chemicals through solid-state fermentation (SSF). A diverse array of microbes is employed in the SSF operations to produce these profitable products. As a result, this study thoroughly examines and discusses the effects of SSF on the production of cost-effective products.

Recycling greenhouse gases from industrial emissions is necessary for a genuine circular carbon economy. One-carbon (C1) compounds like methanol produced from greenhouse gases and its subsequent use as a feedstock hold great promise in driving the next generation of biomanufacturing. This review explores the emerging field of synthetic methylotrophy, which focuses on engineering microbial cell factories to convert methanol into useful bioproducts like industrial chemicals, pharmaceuticals, fuels, and food. Native methylotrophs have natural pathways for methanol utilization, but obstacles such as metabolic inefficiency and the availability of genetic modification tools limit their use. In contrast, Synthetic methylotrophy makes use of model organisms such as Escherichia coli and Saccharomyces cerevisiae, which can be genetically altered to enhance the efficiency of bioconversion and methanol utilization. Although formaldehyde detoxification and enzyme optimization have improved recently due to developments in metabolic engineering, there are still many obstacles to overcome, such as limited methanol uptake and toxicity problems. The recent developments in synthetic methylotrophy are highlighted in this review, which also stresses the necessity of integrating advanced synthetic biology techniques and performing further research into metabolic pathways of methanol assimilation. Together with a consideration of the techno-economic aspects affecting the scalability of these novel processes, the potential for C1-based biomanufacturing to support sustainable production methods is emphasized.

The collection and utilization of CO₂ are recognized as the key strategies for mitigating global climate change. Recently, numerous microorganisms capable of utilizing CO₂ as a carbon source for growth have been explored and engineered for biomanufacturing. However, these processes are typically initiated by exposing microorganisms to high concentrations of CO₂, which significantly limits the application of microbial carbon fixation in synthetic biology. Here, we demonstrate that Komagataella phaffii (K. phaffii) can fix CO₂ via the Reductive Glycine Pathway (RGlyP) at a low concentration (0.5% CO₂). We propose that the endogenous RGlyP in glycine auxotroph K. phaffii A01 can effectively operate under growth pressure and utilize CO₂ to synthesize glycine. The molecular mechanisms involved are elucidated at the transcriptional level. This is the most efficient RGlyP reported so far, demonstrating the great potential of the endogenous RGlyP in K. phaffii for CO₂ fixation and utilization research and will further promote the development of synthetic biology, contributing to the mitigation of global climate and food crises.

α-Farnesene is a valuable sesquiterpene with wide applications in cosmetics, biofuels, and pharmaceuticals. Microbial production of α-farnesene from low-cost carbon sources represents a sustainable alternative to chemical synthesis. In this study, Pichia pastoris was engineered to efficiently produce α-farnesene using methanol as the sole carbon source. First, the native mevalonate pathway was systematically optimized by stepwise overexpression of ERG10, ERG13, a truncated HMG1, and key downstream enzymes, resulting in P. pastoris cfn3 with α-farnesene production up to 172.1 mg/L. Then, adaptive laboratory evolution was conducted to improve methanol tolerance, and the evolved strain P. pastoris evo13-4 grew more rapidly than that of P. pastoris cfn3. To further boost α-farnesene production, ARTP mutagenesis was used to generate numerous mutants for screening strains with high performance, among which P. pastoris ccg3-8 exhibited the highest production of α-farnesene up to 449.4 mg/L. Finally, in a 5-L bioreactor, α-farnesene production with the resulting strain P. pastoris ccg3-8 reached 3.28 g/L. This study presents an effective strategy for engineering P. pastoris for methanol-based biosynthesis of α-farnesene, providing a promising platform for the effective and sustainable production of isoprenoid compounds.

2′-Fucosyllactose is the most abundant human milk oligosaccharides and one of the three essential nutrients for infant growth. Corynebacterium glutamicum is one of the most common industrial fermentation bacteria but cannot synthesize 2′-fucosyllactose. In this study, C. glutamicum ATCC13032 was engineered for 2′-fucosyllactose production from fucose and lactose. The gene futC from Helicobacter pylori encoding α-1,2-fucosyltransferase was codon optimized and mutated at four amino acids (F40S/Q150H/C151R/Q239S). The modified gene futC and the gene fkp from Bacteroides thetaiotaomicron encoding fucokinase/GDP-fucose pyrophosphorylase were overexpressed in plasmid pEC and transformed into C. glutamicum, resulting in CW002. CW002 did not synthesize 2′-fucosyllactose possibly because the substrates fucose and/or lactose did not pass through the cell membrane. Therefore, the gene lacY encoding lactose permease and the gene fucP encoding fucose permease from Escherichia coli were overexpressed in plasmid pXTuf and transformed into CW002, resulting in CW006. CW006 did synthesize 2′-fucosyllactose as expected. It is interesting that the production of 2′-fucosyllactose was decreased or stopped when the expression combination of these four genes was changed, suggesting that the expression levels of the four genes in CW006 might have to well balanced. C. glutamicum CW006 produced 2.07 g/L 2′-fucosyllactose in a 2.4 L bioreactor.

Bacillus natto (B. natto) can produce various secondary metabolites, notably the coagulation-promoting menaquinone-7 (MK-7) and the natural multifunctional biopolymer poly-γ-glutamic acid (γ-PGA). To enhance the economic feasibility of the fermentation process. In this study, the ability to synthesize MK-7 of B. natto ND-1 was enhanced through atmospheric pressure room temperature plasma (ARTP)-induced mutagenesis. Subsequently, we found a significant amount of γ-PGA in the fermentation product. Additionally, the fermentation medium and cultural conditions were rigorously optimized. An optimal medium composed of 70 g/L glycerol, 200 g/L soy peptone, 50 g/L yeast extract, and 0.04 g/L K₂HPO₄ was obtained by Orthogonal experimental design. Following 96 h of liquid-state fermentation without agitation, the concentrations of MK-7 and γ-PGA were 48.31 ± 3.17 mg/L and 92.53 ± 2.71 g/L, respectively. These results suggested that the co-production of MK-7 and γ-PGA has demonstrated bioactivity and stability, providing a theoretical foundation for their potential application in the domains of food, medicine, and nutraceuticals.

In Maotai-flavor Baijiu production, reducing lactic acid (LA) can alleviate microbial imbalance and flavor disharmony caused by LA accumulation. Current methods for reducing LA mainly focus on physical removal and fermentation parameter control, but they address only the symptoms, not the underlying cause. This study selected Lactobacillus panis antagonistic bacteria to control LA production at its source by inhibiting L. panis growth and analyzed its antimicrobial substances and mechanisms. Firstly, a high-throughput screening method for L. panis antagonists was developed based on lactate dehydrogenase, which correlates LA concentration with reduced nicotinamide adenine dinucleotide (NADH). Subsequently, a total of 34 antagonists were screened, with Bacillus licheniformis BL-4 exhibiting the highest inhibition rate (62.25%) against L. panis. Moreover, the primary antimicrobial substance, antimicrobial peptide antiL24, was purified from B. licheniformis BL-4 and evaluated for its activity and sequence. Finally, the mechanism of antiL24 against L. panis was analyzed by using microstructural analyses and transcriptomic profiling, revealing that antiL24 disrupts the cell wall and membrane of L. panis and affects genes involved in energy metabolism and protein synthesis. This study proposes a novel strategy for regulating LA concentration in Maotai-flavor Baijiu production, with the potential to enhance its quality.

Next-generation sequencing technologies have significantly advanced our comprehension of microbial diversity and ecological roles within fermented foods. However, culture-based approaches remain essential for a comprehensive understanding of these complex ecosystems. This study integrated culture-dependent and culture-independent techniques to elucidate the microbial diversity and flavor-forming potential of fungi in high-temperature Daqu, a critical solid-state fermentation process in Maotai-flavor Baijiu production. Through iterative cultivation strategies, we successfully isolated and identified 660 pure eukaryotic colonies, representing 33 genera and 58 species, from Daqu samples. This approach significantly improved cultivation efficiency from 20.7 to 63.2%. In addition, despite the optimization of ITS rRNA primer sets to enhance the detection of eukaryotic microorganisms, we still identified 21 genera (comprising 27 species) that were culturable but not detected by high-throughput sequencing analysis. Fermentation experiments demonstrated the robust growth and substantial ethyl acetate production potential of these fungal species, particularly Kluyveromyces marxianus, which exhibited exceptional performance at elevated temperatures (45 °C). This study advanced culture-dependent techniques to improve the isolation of eukaryotic microorganisms from Daqu. These findings highlight the importance of culture-based approaches in characterizing microbial diversity and function.

Isomaltulose stands out for its low glycemic index and caries-resistant properties, and shows great potential for applications in the food and medical fields. Belonging to the glycoside hydrolase family GH13, sucrose isomerase is capable of converting sucrose to isomaltulose. The sucrose isomerase from Pantoea dispersa UQ68J (PdSI) is favored for its high conversion rates. However, poor thermostability limits its application in industrial production. To enhance the thermostability of PdSI, we combined sequence analysis and computer-aided design to identify and exclude key sites that might affect catalytic activity, and then screened 14 candidate mutants for point mutation validation. During the study, single-point mutants M62E, V105I, N109H, D232P, V447E and S481M demonstrated improved thermostability in preliminary experiments. Among them, the mutant V447E performed particularly well, with a 1.38-fold increase in half-life at 40 °C compared to the wild type, and showed an increase in the optimal temperature from 30 °C to 35 °C. Further combined mutation studies revealed that mutant V447E/D232P showed better thermostability. Compared with the wild type, mutant V447E/D232P increased the optimal temperature by 5 °C, and its half-life at 40 °C was prolonged by 1.52-fold. The results of molecular dynamics simulations further confirmed the low root-mean-square fluctuations of V447E and V447E/D232P compared with the wild type, indicating a significant enhancement in structural stability. This study offers a reference for improving the thermostability modification of sucrose isomerase and promotes the industrial application of isomaltulose.

Alcohol dehydrogenases (ADHs) are key enzymes in microbial ethanol metabolism and ethanol detection with significant relevance in industrial bioprocessing and synthetic biology. The study focuses on enhancing the activation of Magnusiomyces capitatus ADHs specific to ethanol for ethanol conversion and detection. This was achieved by evaluating its specific alcohol dehydrogenase (ADH) activity under varying growth conditions by following a systematic one-factor-at-a-time (OFAT) approach and a central composite rotatable design (CCRD). Using the OFAT method, the most critical factor for improving specific ADH activity were identified as glucose, ammonium sulphate, zinc sulphate, and pH, which was further optimized using the CCRD. The specific ADH activity of M. capitatus in the developed medium was 489.28 ± 0.31 mU mg^− 1 of protein, which was greater than that of cells cultured in basal ethanol medium. Furthermore, the volatile compounds (VOCs) generated during ethanol oxidation under aerobic conditions were analyzed by GC-MS, validating the metabolic flexibility of M. capitatus under optimal circumstances. These findings offer new insights into the systems-level metabolic behavior of M. capitatus under ethanol stress and highlight its potential as a microbial platform for future biomanufacturing and enzymatic conversion processes with potential applications in biosensing and industrial bioprocessing.

Xylan is the major hemicellulose component of the plant cell wall (second most naturally abundant carbohydrate) and is a linear polymer of β-d-xylopyranosyl units linked by β-1-4 glycosidic bonds. Microbial xylanase is an efficient xylan degrading enzyme that catalyzes the hydrolysis of internal β-1-4 glycosidic bonds and is reported to be involved in bioethanol production from lignocellulosic biomass. Due to its wide range of applications at the industrial level, it is important to understand the structural and functional aspects of xylanase. Therefore, in the present study, an in silico investigation was carried out through the docking of bacterial xylanases with multiple substrates xylobiose, xylotriose, xylotetraose, and xylopentaose to determine the molecular interaction and substrate specificity during enzymatic catalysis. The amino acid sequences of four xylanolytic bacterial species, viz., Actinosynnema pretiosum, Streptomyces sp., Spirosoma sordidisoli and Streptomyces bingchenggensis were retrieved from UniProtKB and their homologous structures were predicted using the SWISS-PROT model webserver to undertake docking studies using the xylanase enzyme of the above bacterial species and xylan as a substrate. Results of the docking studies showed that the xylanase of all the bacterial species exhibited the highest interaction with xylopentaose. Binding energy was determined using the DINC server. Further multiple sequence alignment (MEGA X), phylogenetic analysis (MEGA X), and molecular dynamics simulation (GROMACS) studies were performed. Overall, the present in silico study will reveal the importance of understanding the catalytic mechanism of substrate xylan with different bacterial xylanases, which could be helpful for the development of engineered xylanase towards the efficient production of bioethanol from lignocellulosic biomass.

This study reports the development of a novel and cost-effective cellulolytic enzyme cocktail, named Remzyme, using Rasamsonia emersonii. By supplementing the heterologously expressed carbohydrate-active enzymes (CAZymes) such as lytic polysaccharide monooxygenase (Rem_LPMO1, Rem_GH7CBHI), and xylanase (Malci_GH10xyl), the cocktail was optimized using a Simplex lattice mixture design. This innovative blend achieved a saccharification efficiency of 98.59% when applied to unwashed, acid/steam-pretreated rice straw slurry sourced from an industrial-scale 2G ethanol plant. The process was conducted under industrially relevant conditions with 15% substrate loading and protein loading of 8 mg/g dry substrate. Remarkably, the Remzyme cocktails was comparable to the leading commercial enzyme mix, CellicCTec3, at equivalent protein loadings, underscoring its potential as a cost-effective alternative in enzymatic saccharification. The study demonstrates the synergistic efficacy of accessory enzymes and core cellulases, offering significant advancements in enzyme technology for biorefinery applications.

Farnesol (FOH), a prized sesquiterpenoid alcohol, is at the core of this study, which outlines a synthetic biology strategy to significantly boost its production for use in flavors, fragrances, pharmaceuticals, and biofuels. We constructed an efficient FOH biosynthetic pathway in Serratia marcescens, leveraging rational engineering strategies to optimize its production. Initially, we introduced a heterologous mevalonate (MVA) pathway into S. marcescens for FOH biosynthesis. We then screened different sources of monophosphate phosphatases and performed rational modifications to enhance their activity. Computational simulations were employed to model the SmAp-FP complex, guiding protein engineering efforts. The engineered strain S. marcescens SPF6_L2 achieved a FOH titer of 457.3 ± 23.1 mg/L in shake flask fermentation, which was further scaled up to 1784.3 mg/L in a 5 L fermenter. This represents one of the highest reported titers of FOH production in microorganisms to date. Our approach integrates genetic engineering, enzyme optimization, and bioprocess design to efficiently biosynthesize FOH. It sets the stage for future research on optimizing S. marcescens metabolic pathways for enhanced terpenoid biosynthesis.

Microbial cell factories are widely used for the bioproduction of various chemicals and biofuels. During this process, microorganisms will encounter many different stresses that frequently induce oxidative stress, thereby compromising cell growth. Here, Candida glabrata was used as a model system to engineer its oxidative stress tolerance by introducing malate biosynthetic capability. To further improve oxidative stress tolerance, malate biosynthesis pathway was optimized by fine-tuning expression strengths of pyruvate carboxylase and malate dehydrogenase, leading to an enhanced oxidative stress tolerance. Then, the physiological mechanism under this phenomenon was explored, and the antioxidative defense system showed a good improvement in ROS content, intracellular ATP level, superoxide dismutase and catalase activity. Further, the malate-producing C. glabrata was used to analyze their tolerance to artemisinin, showing that C. glabrata tolerance to artemisinin was significantly enhanced by introducing the malate biosynthetic capability. This study presented herein opens a window for the development of efficient cell factories with high tolerance to environment stress, facilitating the biosynthesis of pharmaceutical chemicals.

Monensin, a polyether ionophore antibiotic that is produced by Streptomyces cinnamonensis through fermentation, is extensively utilized in both agricultural and pharmaceutical sectors. This study focused on identifying some specific genes and critical metabolic pathways related to the monensin biosynthesis in S. cinnamonensis for efficient monensin production with a genome analysis. Results show that genes of the strain were significantly enriched in the monensin synthetic pathway, including primary metabolic (central carbon and fatty acids) processes, energy metabolism, and secondary metabolite biosynthesis, which was largely potential in the supply of sufficient building precursors and energy. The annotated specific genes were predominantly located in metabolic pathways and secondary metabolites biosynthesis, accounting for 90.63% and 39.06%, respectively. Among them, specific genes, fadD, fadE, fadB, and fadA in the fatty acid degradation pathway were apparently the most prominent. With single overexpression, these genes resulted in the strain increasing monensin titer by 14%, 11%, 22%, and 10%, respectively. Further, with the tandem overexpression, an engineered strain M5 was successfully constructed. The strain was capable of producing 18.88 g/L of monensin at 288 h at shake-flask level and 37.31 g/L via fed-batch in a 50-L bioreactor, which is 1.3 folds and 1.2 folds, respectively, that of the starting strain. To our knowledge, this represents the highest level reported to date, which is of a big industrial promise. This study provides a genetic foundation for elucidating the monensin synthesis mechanism and paves the way for metabolic engineering modifications and industrial production.

4-Hydroxyisoleucine (4-HIL) holds potential value in the treatment of diabetes. It can be produced by expressing the exogenous isoleucine dioxygenase gene ido in L-isoleucine (Ile) producing Corynebacterium glutamicum strains. But the stable expression of ido on plasmids relies on the usage of antibiotics. To make the harboring of ido independent of plasmid, this study developed a chromosome-engineered strain for synthesizing 4-HIL directly from glucose. First, the ido-cat-ido expressing cassette was inserted into the chromosome of C. glutamicum, and the copy number of ido was increased through chemically inducible chromosome evolution (CIChE). After successive rounds of CIChE by increasing chloramphenicol concentration, 7 copies of ido were integrated in the chromosome of C. glutamicum SE04, and the 4-HIL production reached 20.3 ± 4.99 g/L, 3.5-fold higher than the initial strain SC12 harboring two-copies of ido. To cease further homologous recombination, recA was deleted in CIChE strains, but cell growth and 4-HIL production were damaged. Notably, the stability of chromosomally inserted genes in the evolved strain SE04 was confirmed. Ultimately, the evolved C. glutamicum SE04 strain produced 30.3 g/L of 4-HIL in a 2-L bioreactor. This study established a plasmid-free strain of C. glutamicum for 4-HIL production, offering new insights into utilizing multi-copy integration methods for producing other valuable biochemical substances in C. glutamicum.

Phi29 DNA polymerase (Phi29 Pol) has emerged as a powerful tool in the third-generation sequencing technology such as DNA nanoball-based sequencing. However, natural Phi29 Pol with low amplification activity under high-salt conditions needs to be engineered to meet specific sequencing demands, which are usually achieved through a time-consuming and iterative trial-and-error process. Herein, we develop a high-throughput screening methodology for efficiently detecting Phi29 Pol mutants with high rolling-circle amplification (RCA) efficiency under high-salt conditions. The method uses a nucleic acid gel stain sensitive to oligonucleotides to achieve the input conversion from enzymatic amplification efficiency to fluorescence intensity in micron-sized droplets. We further demonstrate the potential of this methodology in the first high-throughput droplet sorting of Phi29 Pol. The RCA efficiency of sorted mutant S6 is 1.39-fold that of initial enzyme M2 in 300 mM KCl. Overall, this study provides a cost-effective and rapid solution for improving the performance of Phi29 Pol under high-salt conditions.

Efficient methanol assimilation was crucial for methanol-based biomanufacturing of high-valued products. Given the low methanol utility in native Komagataella phaffii cells, we disrupted the genes encoding formaldehyde dehydrogenase and formate dehydrogenase and incorporated heterologous RuMP into K. phaffii to drive more flux into central metabolic pathways. We also performed transcriptome analysis to evaluate the metabolic impact of this genetic modification. The results showed that the biomass of Δfldh mutant strain was 8.3% higher than that of the wild type control strain. The ratio of biomass accumulation from methanol was respectively 5.754 and 6.209 in WT and Δfldh. Recombinant RuMP-Δfdh and hps-Δfdh rescued the unliving Δfdh and were able to growth in medium with methanol as sole carbon source. In addition, the transcription revealed the impact of disrupted fldh or fdh on TCA, PPP and the respiratory chain. Our results suggested that inactivation of fldh and expressing RuMP were beneficial for methanol utility. It was fdh rather than fldh that was indispensable for K. phaffii cell growth. This study provided new insights into how to reprogram K. phaffii to enhance its methanol assimilation rate, also the theoretical bases for the mechanism underlying the better methanol digestion.

When recombinant E. coli BL21(DE3) is induced to express Bacillus subtilis lipase A (BsLipA) using Isopropyl β-D-1-thiogalactopyranoside (IPTG), the one-time addition of IPTG leads to problems such as limited cell growth, a short enzyme production period, and low yield. To address these issues, this study proposes an innovative IPTG feeding strategy, where the IPTG feeding rate is adjusted based on cell growth rate between 4 and 10 h, followed by a constant IPTG feeding rate after 12 h. Fermentation experiments in a 5 L bioreactor demonstrated that IPTG feeding according to this strategy resulted in continuous enhancement of BsLipA activity, reaching 288.50 U/mL. Compared to a batch induced with a one-time addition of 0.2 mmol/L IPTG, BsLipA activity increased by 6.67 times. This IPTG feeding strategy was applied to a low-nutrient fermentation process with DO-start glucose feeding, leading to a further increase in BsLipA enzyme activity, with the highest activity reaching 580.29 U/mL. The results indicate that this strategy significantly reduces the toxic effects of IPTG on the cells, improves biomass, extends the enzyme production phase, and enhances BsLipA expression levels by balancing the induction strength and cell growth conditions.

Media engineering and strain improvement are critical aspects of microbial biotechnology playing a vital role in enhancing microbial productivity, and ensuring cost-effective bioprocessing. In this investigation, we optimized the various medium components, nutritional condition, and fermentation parameters for the industrial production of phenoxymethylpenicillin or penicillin V (PenV). We have isolated, characterized Penicillium rubens BIONCL P45 strain which initially produced 100 mg/L of PenV. Further, optimization using production medium 4 (PM4) comprising lactose, corn steep solids, sodium sulfate, calcium carbonate, and phenoxy acetic acid lead to a significant increase in production, reaching 430 mg/L. Further improvements through response surface methodology (RSM) predicted a production of 646 mg/L, which was experimentally validated at 685 mg/L. Subsequently, mutagenesis studies using UV (ultraviolet) exposure resulted in the UV-65 mutant, which demonstrated a superior performance, achieving 934 mg/L, surpassing the parental strain. ​These combined strategies lead to a tenfold increase in PenV titer, highlighting their effectiveness in bioprocess development and industrial-scale antibiotic production.

Innovative strategies for sustainable utilization of waste resources are imperative in the pursuit of a circular economy. Recently, the idea of utilizing mammalian spent media as a valuable resource is gaining traction, offering significant opportunities for innovative uses as a food-grade feedstock for microbial fermentation, especially in the production of alternative proteins for research and food purposes. In this study, we aim to repurpose spent mammalian culture media for production of valuable proteins. Growth factors (GFs) are a family of high-value proteins that naturally stimulate cell proliferation or differentiation. More importantly, these factors also present significant costs for cell culture. Here, we successfully demonstrate the use of spent mammalian culture media for the recombinant production of fibroblast growth factor 2 (FGF2-G3) in Lactococcus lactis. Bioreactor fermentation at a 1 L scale confirmed purified yields of 2.6 mg/L of recombinant FGF2-G3 using spent media. Further functional testing indicated that the recombinant FGF2-G3 can promote cell proliferation on an Anguilla japonica (Japanese eel) pre-adipocytic cell line, suggesting its potential for cultivated meat production. Based on the preliminary results of this study, our calculations indicate that fermenting 1 L spent mammalian waste could yield enough growth factors to efficiently grow approximately 52 L of cultivated meat through fermentation. This prediction highlights the potential of waste valorisation to produce reagents for cultivated meats sustainably, thereby contributing to environmental preservation and economic viability.