Yarrowia lipolytica is a promising host for producing valuable chemicals owing to its robustness and metabolic versatility. Efficient genome editing tools are essential for advancing its biotechnological applications. Although CRISPR/Cas9 technology has been applied in Y. lipolytica, achieving a consistently high editing performance remains challenging owing to the low homologous recombination efficiency and variability in system components. In this study, we optimized CRISPR/Cas9-mediated genome editing in Y. lipolytica to enhance its editing efficiency. Using the RNA polymerase III promoter SCR1-tRNA for sgRNA expression, we achieved a gene disruption efficiency of 92.5 %. The tRNA-sgRNA architecture enabled a dual gene disruption efficiency of 57.5 %. KU70 deletion in the Cas9 system increased the integration efficiency to 92.5 %, and Rad52 and Sae2 overexpression boosted homologous recombination. The introduction of Cas9^{D147Y, P411T} (iCas9) enhanced the efficiency of both gene disruption and genome integration. This study provides a powerful tool for efficient gene editing in Y. lipolytica, which will accelerate the construction of yeast cell factories.

In recent years, industrial activities have significantly increased atmospheric CO₂ levels, exacerbating global warming. Carbon reduction involves implementing measures to minimize CO₂ emissions from human activities and achieve a balance between carbon absorption and emissions. Therefore, effective reduction of CO₂ emissions is crucial. Conventional physical and chemical methods for CO₂ fixation frequently cause secondary environmental pollution. As a result, utilizing microorganisms for CO₂ fixation has gained considerable interest. This review provides an overview of the natural pathways for microbial CO₂ fixation, recent advancements in artificial CO₂ fixation, and strategies for enhancing the efficiency of microbial CO₂ fixation. We also discuss the conversion of CO₂ into diverse metabolic products and high-value chemicals. By identifying efficient carbon fixation pathways for microorganisms, this review aims to lay the foundation for the biological production of high-value chemicals using CO₂ as a raw material.

Investigating ecological interactions within microbial ecosystems is essential for enhancing our comprehension of key ecological issues, such as community stability, keystone species identification, and the manipulation of community structures. However, exploring these interactions proves challenging within complex natural ecosystems. With advances in synthetic biology, the design of synthetic microbial ecosystems has received increasing attention due to their reduced complexity and enhanced controllability. Various ecological relationships, including commensalism, amensalism, mutualism, competition, and predation have been established within synthetic ecosystems. These relationships are often context-dependent and shaped by physical and chemical environmental factors, as well as by interacting populations and surrounding species. This review consolidates current knowledge of synthetic microbial ecosystems and factors influencing their ecological dynamics. A deeper understanding of how these ecosystems function and respond to different variables will advance our understanding of microbial-community interactions.

Pentalenolactone is a sesquiterpene antibiotic from Streptomyces. Its biosynthetic pathway has been elucidated, except for the oxidation of pentalen-13-al to 1-deoxypentalenic acid. In this study, we show that cytochrome P450 pentalenene oxygenase catalyzed the formation of 1-deoxypentalenic acid. Ferredoxin XNR_5179 and ferredoxin reductase XNR_4478 from S. albus are suitable redox proteins for pentalenene oxygenase. The biosynthetic pathway presented fills a gap in the biosynthetic pathway of pentalenolactone and provides an example of cytochrome P450 enzyme activity being affected by redox proteins.

Conventional heme enzymes utilize iron-oxygen intermediates to activate substrates and drive reactions. Recently, Chen et al. discovered a novel NADPH-independent superoxide mechanism of heme catalase EasC, which facilitates an O₂-dependent radical oxidative cyclization reaction during ergot alkaloid biosynthesis. This enzyme coordinates superoxide-mediated catalysis by connecting spatially distinct NADPH-binding pocket and heme pocket via a slender tunnel, offering a novel perspective on the catalytic mechanisms of heme enzymes in nature.

Bacteria employ diverse immune systems, such as CRISPR-Cas, to fend off phage infections. A recent study uncovered the unprecedented mechanistic features of the Kongming bacterial defense system, which uniquely exploits phage-derived enzymes to synthesize deoxyinosine triphosphate (dITP), thereby triggering host immunity through NAD+ depletion. In response, some phages have evolved countermeasures to disrupt dITP synthesis, highlighting the ongoing evolutionary arms race between hosts and pathogens. This discovery not only deepens our understanding of bacterial defense strategies but also paves the way for new insights in biomedical research and synthetic biology.

Despite the isolation of over 1000 known bioactive lichen mycobiont-derived secondary metabolites (SMs), understanding the genetic basis of their biosynthesis remains elusive. Biosynthetic gene clusters (BGCs) have been tentatively linked to chemical structures, with core genes such as polyketide synthases (PKSs) surrounded by accessory genes like decarboxylases. In this study, we focused on a decarboxylase gene from the genome of the lichen cladonia uncialis (named as Cu-decarboxylase) to elucidate its role in SM biosynthesis. A 963 bp gene was cloned from C. uncialis and expressed in Escherichia coli (BL21(DE3) cells using the pQE80L expression vector. The resulting 35 kDa protein was purified by applying a Ni⁺-NTA column using an FPLC system. Functional activity assays revealed the decarboxylation and reversible carboxylation of resorcinol to 2,4-dihydroxybenzoic acid and orcinol to orsellinic acid. This suggests a potential role for this Cu-decarboxylase in SM biosynthesis.

Furthermore, the lack of activity on substrates like anthranilic acid and aniline highlighted the importance of the phenolic OH group in facilitating these reactions. The 3D protein structure was predicted with AlphaFold3, based on sequence similarity with a known decarboxylases and revealed the importance of a zinc cofactor for the catalytic activity of the enzyme. The optimization of the reaction conditions, particularly for orsellinic acid production from orcinol, may enhance conversion rates and offer a viable route for industrial-scale production of bioactive compounds. This study marks the first known instance of functional heterologous expression of a non-codon-optimized gene isolated from lichen in E. coli.

Efficient conversion of corn stover to bioethanol via simultaneous saccharification and fermentation (SSF) is a promising strategy for sustainable biofuel production. A major current barrier to this process is the limited thermotolerance of Saccharomyces cerevisiae, which hampers its performance under the high-temperature conditions required for efficient SSF. In this study, we identified TrRCC1, a gene from Trichoderma reesei, as a candidate for improving microbial stress resistance. Overexpression of TrRCC1 in both T. reesei Rut C30 and S. cerevisiae BY4741 significantly enhanced thermotolerance. In T. reesei Rut C30, TrRCC1 overexpression improved heat resistance and increased cellulase production by 2.5-fold compared to the wild-type strain. In S. cerevisiae BY4741, TrRCC1 overexpression resulted in enhanced thermotolerance and a 21.8 % increase in ethanol production during SSF of corn stover. The ethanol concentration achieved in the SSF process with TrRCC1-overexpressing S. cerevisiae was 44.1 g/L, which was a notable improvement over control strain production. These findings highlight the potential of TrRCC1 as a key gene for engineering microbial strains with improved stress resistance to enhance the efficiency of bioethanol production from lignocellulosic biomass.