Riboflavin (vitamin B₂) is one of the essential vitamins that the human body needs to maintain normal metabolism. Its biosynthesis has become one of the successful models for gradual replacement of traditional chemical production routes. B. subtilis is characterized by its short fermentation time and high yield, which shows a huge competitive advantage in microbial fermentation for production of riboflavin. This review summarized the advancements of regulation on riboflavin production as well as the synthesis of two precursors of ribulose-5-phosphate riboflavin (Ru5P) and guanosine 5′-triphosphate (GTP) in B. subtilis. The different strategies to improve production of riboflavin by metabolic engineering were also reviewed.

Autolysis is a widespread phenomenon in bacteria. In batch fermentation of Clostridium acetobutylicum ATCC 824, there is a spontaneous large-scale autolysis phenomenon with significant decrease of cell density immediately after exponential phase. To unravel the role of autolysis, an autolysin-coding gene, CA_C0554, was disrupted by using ClosTron system to obtain the mutant C. acetobutylicum lyc::int(72). The lower final cell density and faster cell density decrease rate of C. acetobutylicum ATCC 824 than those of C. acetobutylicum lyc::int(72) indicates that CA_C0554 was an important but not the sole autolysin-coding gene responding for the large-scale autolysis. Similar glucose utilization and solvents production but obvious lower cell density of C. acetobutylicum ATCC 824 comparing to C. acetobutylicum lyc::int(72) suggests that lysed C. acetobutylicum ATCC 824 cells were metabolic inactive. On the contrary, the spore density of C. acetobutylicum ATCC 824 is 26.1% higher than that of C. acetobutylicum lyc::int(72) in the final culture broth of batch fermentation. We speculated that spontaneous autolysis of metabolic-inactive cells provided nutrients for the sporulating cells. The present study suggests that one important biological role of spontaneous large-scale autolysis in C. acetobutylicum ATCC 824 batch fermentation is contributing to generation of more spores during sporulation.

Acetogenic bacteria (acetogens) are a class of microorganisms with conserved Wood-Ljungdahl pathway that can utilize CO and CO₂/H₂ as carbon source for autotrophic growth and convert these substrates to acetate and ethanol. Acetogens have great potential for the sustainable production of biofuels and bulk biochemicals using C1 gases (CO and CO₂) from industrial syngas and waste gases, which play an important role in achieving carbon neutrality. In recent years, with the development and improvement of gene editing methods, the metabolic engineering of acetogens is making rapid progress. With introduction of heterogeneous metabolic pathways, acetogens can improve the production capacity of native products or obtain the ability to synthesize non-native products. This paper reviews the recent application of metabolic engineering in acetogens. In addition, the challenges of metabolic engineering in acetogens are indicated, and strategies to address these challenges are also discussed.

Acetic and formic acid are two common inhibitors that coexist with glucose and xylose in lignocellulosic hydrolysates, which impair the fermentation performance of Saccharomyces cerevisiae. Enhancing yeast tolerance to these inhibitors is crucial for efficient industrial bioethanol production. Previous transcriptomic studies have indicated the involvement of the transcription factors Haa1p and Hap4p in the cellular response to mixed acetic and formic acid stress. This study aimed to further elucidate their regulatory roles in conferring tolerance to this combined stress condition. Comparative transcriptomic analysis was conducted using the engineered strains s6H3 (HAA1-overexpressing) and s6P5 (HAP4-overexpressing), in comparison with the original strain s6. Both HAA1 and HAP4 overexpression improved fermentation performance, both in the presence and absence of inhibitors. HAA1 overexpression led to a greater number of differentially expressed genes (DEGs) under mixed acid stress compared to non-inhibitory conditions. Genes involved in glycolysis, the pentose phosphate pathway (PPP), necroptosis, and ribosome biogenesis were significantly downregulated, whereas those associated with the glyoxylate cycle, nucleotide metabolism, and RNA polymerase activity were significantly upregulated. In contrast, HAP4 overexpression resulted in fewer DEGs under acid stress conditions, which may be attributed to the intrinsic induction of HAP4 in the original strain s6 under acid exposure. Under these conditions, genes related to metabolic regulation, RNA processing, and transcription were significantly downregulated, while those involved in transport, ribosome biogenesis, genome stability, and sporulation were significantly upregulated. Collectively, both Haa1p and Hap4p appear to regulate other transcription factors, thereby indirectly influencing global gene expression in response to mixed acetic and formic acid stress. This study provides the experimental evidence for the protective role of Haa1p and Hap4p under combined acetic and formic acid stress. Regulatory mechanisms underlying the responses of Haa1p and Hap4p to combined acid stress were identified, expanding current understanding of yeast stress adaptation.

Clostridium ljungdahlii is a model acetogenic bacterium utilized for ethanol production from syngas, with its growth and ethanol synthesis being profoundly influenced by fermentation pH. However, the mechanistic basis of this pH-dependent regulation remains poorly understood. In this study, we systematically investigated the impact of pH on the growth and metabolic profile of C. ljungdahlii under controlled pH conditions using CO as the sole carbon and energy source. At pH 6.0, C. ljungdahlii consumed around 6.0 M carbon monoxide, producing 413 ± 43 mM acetate, 288 ± 35 mM ethanol, and 17 ± 2 mM 2,3-butanediol, with a maximum optical density (OD) of 15.9. In contrast, at pH 5.3, the strain exhibited enhanced metabolic activity, consuming around 9.6 M carbon monoxide and generating 235 ± 24 mM acetate, 756 ± 26 mM ethanol, 38 ± 4 mM 2,3-butanediol, and 28 ± 7 mM lactate, achieving a maximum OD of 30. This represents an approximate twofold increase in both ethanol production and biomass accumulation compared to pH 6.0. Proteomic and parallel reaction monitoring (PRM) analyses demonstrated that the expression levels of key enzymes in central metabolic pathways were marginally higher at pH 6.0 than at pH 5.3, indicating that the observed physiological enhancements were not attributable to differential enzyme expression but likely stemmed from variations in ATP synthesis efficiency. Further optimization experiments revealed that the optimal pH for growth and ethanol production by C. ljungdahlii under CO-sufficient and nutrient-replete conditions is approximately 5.3. These findings provide critical insights into the pH-dependent metabolic regulation of C. ljungdahlii and establish essential parameters for scaling up syngas fermentation for ethanol production. Additionally, this study offers a foundation for further exploration of the unique proton motive force-driven ATP synthesis system in C. ljungdahlii and its broader implications for metabolic network regulation.

This study investigates the structural effects of dilute acid and alkali treatments on cotton fibers, aiming to understand the influence of chemical pretreatment on cellulose morphology. Cotton samples were exposed to 1% sulfuric acid and 1% sodium hydroxide at 90 °C, and the resulting changes were evaluated using scanning electron microscopy and nonlinear optical imaging techniques. The results indicate that sulfuric acid causes significant fiber degradation, leading to fragmentation and reduced fiber thickness. In contrast, sodium hydroxide treatment results in a roughened, flaky surface while preserving the overall structural integrity, with fibers appearing fluffier and more accessible to enzymatic processes. Untreated cotton fibers maintained a smooth and uniform surface, confirming the chemical specificity of the observed changes. These findings are crucial for optimizing biomass pretreatment methods, demonstrating that dilute chemical treatments primarily affect macrostructural features without significantly disrupting the cellulose microfibrils. The study provides valuable insights for the development of efficient biorefining processes and sustainable bio-based materials, highlighting the importance of selecting appropriate chemical conditions to enhance enzymatic hydrolysis and biomass conversion while maintaining the core structure of cellulose. This research contributes to advancing the understanding of cellulose’s structural resilience under mild chemical pretreatment conditions.