This research identified four amino acid residues (Leu174, Asn297, Tyr301, and Gln291) that contribute to substrate recognition by the high-affinity glucose transporter Xltr1p from Trichoderma reesei. Potential hotspots affecting substrate specificity were selected through homology modeling, evolutionary conservation analyses, and substrate-docking modeling of Xltr1p. Variants carrying mutations at these hotspots were subsequently obtained via in silico screening. Replacement of Leu174 or Asn297 in Xltr1p with alanine resulted in loss of hexose transport activity, indicating that Leu174 and Asn297 play essential roles in hexose transport. The Y301W variant exhibited accelerated mannose transport, but lost galactose transport capacity, and mutation of Gln291 to alanine greatly accelerated mannose transport. These results suggest that amino acids located in transmembrane α-helix 7 (Asn297, Tyr301, and Gln291) play critical roles in substrate recognition by the hexose transporter Xltr1p. Our results will help expand the potential applications of this transporter and provide insights into the mechanisms underlying its function and specificity.

Cytochrome P450 enzymes (CYPs or P450s) and ferredoxins (Fdxs) are ubiquitously distributed in all domains of life. Bacterial P450s are capable of catalyzing various oxidative reactions with two electrons usually donated by Fdxs. Particularly in Streptomyces, there are abundant P450s that have exhibited outstanding biosynthetic capacity of bioactive metabolites and great potential for xenobiotic metabolisms. However, no systematic study has been conducted on physiological functions of the whole cytochrome P450 complement (CYPome) and ferredoxin complement (Fdxome) of any Streptomyces strain to date, leaving a significant knowledge gap in microbial functional genomics. Herein, we functionally analyze the whole CYPome and Fdxome of Streptomyces venezuelae ATCC 15439 by investigating groups of single and sequential P450 deletion mutants, single P450 overexpression mutants, and Fdx gene deletion or repression mutants. Construction of an unprecedented P450-null mutant strain indicates that none of P450 genes are essential for S. venezuelae in maintaining its survival and normal morphology. The non-housekeeping Fdx1 and housekeeping Fdx3 not only jointly support the cellular activity of the prototypic P450 enzyme PikC, but also play significant regulatory functions. These findings significantly advance the understandings of the native functionality of P450s and Fdxs as well as their cellular interactions.

Inflammatory bowel disease (IBD) is a chronic and recurrent disease caused by immune response disorders that disrupt the intestinal lumen symbiotic ecosystem and dysregulate mucosal immune functions. Current therapies available for IBD primarily focus on symptom management, making early diagnosis and prompt intervention challenging. The development of genetically engineered bacteria using synthetic biology presents a new strategy for addressing these challenges. In this review, we present recent breakthroughs in the field of engineered bacteria for the treatment and detection of IBD and describe how bacteria can be genetically modified to produce therapeutic molecules or execute diagnostic functions. In particular, we discuss the challenges faced in translating live bacterial therapeutics from bacterial design to delivery strategies for further clinical applications.

The depletion of fossil fuels and their impact on the environment have led to efforts to develop alternative sustainable fuels. While biofuel derived from lignocellulose is considered a sustainable, renewable, and green energy source, enhancing biofuel production and achieving a cost-effective bioconversion of lignocellulose at existing bio-refineries remains a challenge. Consolidated bioprocessing (CBP) using thermophiles can simplify this operation by integrating multiple processes, such as hydrolytic enzyme production, lignocellulose degradation, biofuel fermentation, and product distillation. This paper reviews recent developments in the conversion of lignocellulose to biofuel using thermophile-based CBP. First, advances in thermostable enzyme and thermophilic lignocellulolytic microorganism discovery and development for lignocellulosic biorefinery use are outlined. Then, several thermophilic CBP candidates and thermophilic microbes engineered to drive CBP of lignocellulose are reviewed. CRISPR/Cas-based genome editing tools developed for thermophiles are also highlighted. The potential applications of the Design-Build-Test-Learn (DBTL) synthetic biology strategy for designing and constructing thermophilic CBP hosts are also discussed in detail. Overall, this review illustrates how to develop highly sophisticated thermophilic CBP hosts for use in lignocellulosic biorefinery applications.

Recently, Chen et al. published their breakthrough results on a marine microbial genomic catalog and genetic potentials in bioprospecting in Nature, providing unprecedented opportunities for development and utilization of genetic resources of marine microorganisms. To highlight this article, we summarized and highlighted their breakthroughs seriatim

Surface adsorption is a convenient and readily available method for immobilizing enzymes on metal-organic frameworks (MOFs). Metal-organic framework-5 (MOF-5), isoreticular metal-organic frameworks-3 (IRMOF-3), and multivariate analysis of MOF-5/IRMOF-3 (MMI) with a half-amino group (-NH₂) were prepared in this study. Thermomyces lanuginosus lipase (TLL) was chosen as a commercially available enzyme for immobilization on the surfaces of these MOFs. Briefly, 1.5 mg of TLL was added to 10 mg of the MOFs, and after 24 h, 67, 74, and 88% of the TLL was immobilized on MOF-5, IRMOF-3, and MMI, respectively. Fourier transform infrared spectroscopy, X-ray diffraction, thermogravimetric analysis, scanning electron microscopy, energy-dispersive X-ray analysis, and Brunauer-Emmett-Teller analysis were used to characterize the resulting biocomposites. TLL@MOF-5, TLL@IRMOF-3, and TLL@MMI exhibited activities of 55, 75, and 110 U/mg, respectively. Investigation of the activity and stability of the prepared biocatalysts showed that TLL immobilized on MMI was 2.34-fold more active than free TLL. TLL@MMI exhibited high stability and activity even under harsh conditions. After 24 h of incubation in a mixture of 50% (v/v) MeOH, TLL@MMI retained 80% of its activity, whereas TLL@MOF-5 and free TLL lost 50 and 60% of their activities, respectively. TLL@MMI was used to synthesize 2-arylidenehydrazinyl-4-arylthiozole derivatives (91-98%) in a one-pot vessel by adding benzaldehydes, phenacyl bromides, and thiosemicarbazide to water. The efficiency of the 4a derivative with free TLL was 43%, whereas that with TLL@MMI was 98%.

Diverse microbial community structures (MCS) in wastewater treatment plants (WWTPs) are vital for effectively removing nutrients and chemicals from wastewater. However, the regular monitoring of MCS in WWTP bioreactors remains unattractive owing to the skill and cost required for deploying modern microbial molecular techniques in the routine assessment of engineered systems. In contrast, low-resolution methods for assessing broad changes in the MCS, such as phospholipid fatty acid (PLFA) analysis, have been used effectively in soil studies for decades. Despite using PLFA analysis in soil remediation studies to capture the long-term effects of environmental changes on MCS, its application in WWTPs, where the microbial mass is dynamic and operational conditions are more fluid, remains limited. In this study, microbial communities in a controlled pilot plant and 12 full-scale activated sludge plants (ASPs) were surveyed over a two-year period using PLFA analysis. This study revealed that changes in the MCS in wastewater bioreactors could be detected using PLFA analysis. The MCS comprised 59 % Gram-negative and 9 % Gram-positive bacteria, 31 % fungi, and 1 % actinomycetes. The abundances of Gram-negative bacteria and fungi were strongly inversely correlated, with an R²=0.93, while the fatty acids cy17:0 and 16:1ω7c positively correlated (R²=0.869). Variations in temperature, solid retention time, and WWTP configuration significantly influenced the MCS in activated sludge reactors. This study showed that WWTP bioreactors can be routinely monitored using PLFA analysis, and changes in the bioreactor profile that may indicate imminent bioreactor failure can be identified.

Interspecific interactions are an important component and a strong selective force in microbial communities. Over the past few decades, there has been a growing awareness of the variability in microbial interactions, and various studies are already unraveling the inner working dynamics in microbial communities. This has prompted scientists to develop novel techniques for characterizing the varying interspecific interactions among microbes. Here, we review the precise definitions of pairwise and high-order interactions, summarize the key concepts related to interaction variability, and discuss the strengths and weaknesses of emerging characterization techniques. Specifically, we found that most methods can accurately predict or provide direct information about microbial pairwise interactions. However, some of these methods inevitably mask the underlying high-order interactions in the microbial community. Making reasonable assumptions and choosing a characterization method to explore varying microbial interactions should allow us to better understand and engineer dynamic microbial systems.