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Abstract Background Silk proteins have emerged as versatile biomaterials with unique chemical and physical properties, making them appealing for various applications. Among them, spider silk, known for its exceptional mechanical strength, has attracted considerable attention. Recombinant production of spider silk represents the most promising route towards its scaled production; however, challenges persist within the upstream optimization of host organisms, including toxicity and low yields. The high cost of downstream cell lysis and protein purification is an additional barrier preventing the widespread production and use of spider silk proteins. Gram-positive bacteria represent an attractive, but underexplored, microbial chassis that may enable a reduction in the cost and difficulty of recombinant silk production through attributes that include, superior secretory capabilities, frequent GRAS status, and previously established use in industry. Results In this study, we explore the potential of gram-positive hosts by engineering the first production and secretion of recombinant spider silk in the Bacillus genus. Using an industrially relevant B. megaterium host, it was found that the Sec secretion pathway enables secretory production of silk, however, the choice of signal sequence plays a vital role in successful secretion. Attempts at increasing secreted titers revealed that multiple translation initiation sites in tandem do not significantly impact silk production levels, contrary to previous findings for other gram-positive hosts and recombinant proteins. Notwithstanding, targeted amino acid supplementation in minimal media was found to increase production by 135% relative to both rich media and unaltered minimal media, yielding secretory titers of approximately 100 mg/L in flask cultures. Conclusion It is hypothesized that the supplementation strategy addressed metabolic bottlenecks, specifically depletion of ATP and NADPH within the central metabolism, that were previously observed for an E. coli host producing the same recombinant silk construct. Furthermore, this study supports the hypothesis that secretion mitigates the toxicity of the produced silk protein on the host organism and enhances host performance in glucose-based minimal media. While promising, future research is warranted to understand metabolic changes more precisely in the Bacillus host system in response to silk production, optimize signal sequences and promoter strengths, investigate the mechanisms behind the effect of tandem translation initiation sites, and evaluate the performance of this system within a bioreactor.

Cordyceps militaris, a fungus widely used in traditional Chinese medicine and pharmacology, is recognized for its abundant bioactive compounds, including cordycepin and carotenoids. The growth, development, and metabolite production in various fungi are influenced by the complex interactions between regulatory cascades and light-signaling pathways. However, the mechanisms of gene regulation in response to light exposure in C. militaris remain largely unexplored. This study aimed to identify light-responsive genes and potential transcription factors (TFs) in C. militaris through an integrative transcriptome analysis. To achieve this, we reconstructed an expanded gene regulatory network (eGRN) comprising 507 TFs and 8662 regulated genes using both interolog-based and homolog-based methods to build the protein–protein interaction network. Aspergillus nidulans and Neurospora crassa were chosen as templates due to their relevance as fungal models and the extensive study of their light-responsive mechanisms. By utilizing the eGRN as a framework for comparing transcriptomic responses between light-exposure and dark conditions, we identified five key TFs—homeobox TF (CCM_07504), FlbC (CCM_04849), FlbB (CCM_01128), C6 zinc finger TF (CCM_05172), and mcrA (CCM_06477)—along with ten regulated genes within the light-responsive subnetwork. These TFs and regulated genes are likely crucial for the growth, development, and secondary metabolite production in C. militaris. Moreover, molecular docking analysis revealed that two novel TFs, CCM_05727 and CCM_06992, exhibit strong binding affinities and favorable docking scores with the primary light-responsive protein CmWC-1, suggesting their potential roles in light signaling pathways. This information provides an important functional interactive network for future studies on global transcriptional regulation in C. militaris and related fungi.

Abstract Background The increasing prevalence of plastic waste combined with the inefficiencies of mechanical recycling has inspired interest in processes that can convert these waste streams into value-added biomaterials. To date, the microbial conversion of plastic substrates into biomaterials has been predominantly limited to polyhydroxyalkanoates production. Expanding the capabilities of these microbial conversion platforms to include a greater diversity of products generated from plastic waste streams can serve to promote the adoption of these technologies at a larger scale and encourage a more sustainable materials economy. Results Herein, we report the development of a new strain of Pseudomonas bacteria capable of converting depolymerized polyethylene into high value bespoke recombinant protein products. Using hexadecane, a proxy for depolymerized polyethylene, as a sole carbon nutrient source, we optimized media compositions that facilitate robust biomass growth above 1 × 109 cfu/ml, with results suggesting the benefits of lower hydrocarbon concentrations and the use of NH4Cl as a nitrogen source. We genomically integrated recombinant genes for green fluorescent protein and spider dragline-inspired silk protein, and we showed their expression in Pseudomonas aeruginosa, reaching titers of approximately 10 mg/L when hexadecane was used as the sole carbon source. Lastly, we demonstrated that chemically depolymerized polyethylene, comprised of a mixture of branched and unbranched alkanes, could be converted into silk protein by Pseudomonas aeruginosa at titers of 11.3 ± 1.1 mg/L. Conclusion This work demonstrates a microbial platform for the conversion of a both alkanes and plastic-derived substrates to recombinant, protein-based materials. The findings in this work can serve as a basis for future endeavors seeking to upcycle recalcitrant plastic wastes into value-added recombinant proteins.

Spider silk proteins (spidroins) are a remarkable class of biomaterials that exhibit a unique combination of high-value attributes and can be processed into numerous morphologies for targeted applications in diverse fields. Recombinant production of spidroins represents the most promising route towards establishing the industrial production of the material, however, recombinant spider silk production suffers from fundamental difficulties that includes low titers, plasmid instability, and translational inefficiencies. In this work, we sought to gain a deeper understanding of upstream bottlenecks that exist in the field through the production of a panel of systematically varied spidroin sequences in multiple E. coli strains. A restriction on basal expression and specific genetic mutations related to stress responses were identified as primary factors that facilitated higher titers of the recombinant silk constructs. Using these findings, a novel strain of E. coli was created that produces recombinant silk constructs at levels 4–33 times higher than standard BL21(DE3). However, these findings did not extend to a similar recombinant protein, an elastin-like peptide. It was found that the recombinant silk proteins, but not the elastin-like peptide, exert toxicity on the E. coli host system, possibly through their high degree of intrinsic disorder. Along with strain engineering, a bioprocess design that utilizes longer culturing times and attenuated induction was found to raise recombinant silk titers by seven-fold and mitigate toxicity. Targeted alteration to the primary sequence of the recombinant silk constructs was also found to mitigate toxicity. These findings identify multiple points of focus for future work seeking to further optimize the recombinant production of silk proteins and is the first work to identify the intrinsic disorder and subsequent toxicity of certain spidroin constructs as a primary factor related to the difficulties of production.

Abstract Particulate respirators such as N95s are an essential component of personal protective equipment (PPE) for front-line workers. This study describes a rapid and effective UVC irradiation system that would facilitate the safe re-use of N95 respirators and provides supporting information for deploying UVC for decontamination of SARS-CoV-2 during the COVID-19 pandemic. To assess the inactivation potential of the proposed UVC germicidal device as a function of time by using 3 M 8211-N95 particulate respirators inoculated with SARS-CoV-2. A germicidal UVC device to deliver tailored UVC dose was developed and test coupons (2.5 cm2) of the 3 M-N95 respirator were inoculated with 106 plaque-forming units (PFU) of SARS-CoV-2 and were UV irradiated. Different exposure times were tested (0–164 s) by fixing the distance between the lamp and the test coupon to 15.2 cm while providing an exposure of at least 5.43 mWcm−2. Primary measure of outcome was titration of infectious virus recovered from virus-inoculated respirator test coupons after UVC exposure. Other measures included the method validation of the irradiation protocol, using lentiviruses (biosafety level-2 agent) and establishment of the germicidal UVC exposure protocol. An average of 4.38 × 103 PFU ml−1 (SD 772.68) was recovered from untreated test coupons while 4.44 × 102 PFU ml−1 (SD 203.67), 4.00 × 102 PFU ml−1 (SD 115.47), 1.56 × 102 PFU ml−1 (SD 76.98) and 4.44 × 101 PFU ml−1 (SD 76.98) was recovered in exposures 2, 6, 18 and 54 s per side respectively. The germicidal device output and positioning was monitored and a minimum output of 5.43 mW cm−2 was maintained. Infectious SARS-CoV-2 was not detected by plaque assays (minimal level of detection is 67 PFU ml−1) on N95 respirator test coupons when irradiated for 120 s per side or longer suggesting 3.5 log reduction in 240 s of irradiation, 1.3 J cm−2. A scalable germicidal UVC device to deliver tailored UVC dose for rapid decontamination of SARS-CoV-2 was developed. UVC germicidal irradiation of N95 test coupons inoculated with SARS-CoV-2 for 120 s per side resulted in 3.5 log reduction of virus. These data support the reuse of N95 particle-filtrate apparatus upon irradiation with UVC and supports use of UVC-based decontamination of SARS-CoV-2 during the COVID-19 pandemic.

Abstract Background Product toxicity is one of the bottlenecks for microbial production of biofuels, and transporter-mediated biofuel secretion offers a promising strategy to solve this problem. As a robust microbial host for industrial-scale production of biofuels, Saccharomyces cerevisiae contains a powerful transport system to export a wide range of toxic compounds to sustain survival. The aim of this study is to improve the secretion and production of the hydrophobic product (β-carotene) by harnessing endogenous ABC transporters combined with physiological engineering in S. cerevisiae. Results Substrate inducibility is a prominent characteristic of most endogenous transporters. Through comparative proteomic analysis and transcriptional confirmation, we identified five potential ABC transporters (Pdr5p, Pdr10p, Snq2p, Yor1p, and Yol075cp) for β-carotene efflux. The accumulation of β-carotene also affects cell physiology in various aspects, including energy metabolism, mitochondrial translation, lipid metabolism, ergosterol biosynthetic process, and cell wall synthesis. Here, we adopted an inducible GAL promoter to overexpress candidate transporters and enhanced the secretion and intracellular production of β-carotene, in which Snq2p showed the best performance (a 4.04-fold and a 1.33-fold increase compared with its parental strain YBX-01, respectively). To further promote efflux capacity, two strategies of increasing ATP supply and improving membrane fluidity were following adopted. A 5.80-fold increase of β-carotene secretion and a 1.71-fold increase of the intracellular β-carotene production were consequently achieved in the engineered strain YBX-20 compared with the parental strain YBX-01. Conclusions Overall, our results showcase that engineering endogenous plasma membrane ABC transporters is a promising approach for hydrophobic product efflux in S. cerevisiae. We also highlight the importance of improving cell physiology to enhance the efficiency of ABC transporters, especially energy status and cell membrane properties.

Abstract N-glycolyl chondroitin (Gc-CN) is a metabolite of N -glycolylneuraminic acid (Neu5Gc), a sialic acid that is commonly found in mammals, but not humans. Humans can incorporate exogenous Neu5Gc into their tissues from eating red meat. Neu5Gc cannot be biosynthesized by humans due to an evolutionary mutation and has been implicated in causing inflammation causing human diseases, such as cancer. The study Neu5Gc is important in evolutionary biology and the development of potential cancer biomarkers. Unfortunately, there are several limitations to detecting Neu5Gc. The elimination of Neu5Gc involves a degradative pathway leading to the incorporation of N-glycolyl groups into glycosaminoglycans (GAGs), such as Gc-CN. Gc-CN has been found in humans and in animals including mice, lamb and chimpanzees. Here, we present the biosynthesis of Gc-CN in bacteria by feeding chemically synthesized N-glycolylglucosamine to Escherichia coli. A metabolically engineered strain of E. coli K4, fed with glucose supplemented with GlcNGc, converted it to N-glycolylgalactosamine (GalNGc) that could then be utilized as a substrate in the chondroitin biosynthetic pathway. The final product, Gc-CN was converted to disaccharides using chondroitin lyase ABC and analyzed by liquid chromatography–tandem mass spectrometry with multiple reaction monitoring detection. This analysis showed the incorporation of GalNGc into the backbone of the chondroitin oligosaccharide.

The Agrobacterium-mediated transient gene expression system is a rapid and efficient method for heterologous recombinant protein expression in plants. The fermentation of genetically modified Agrobacterium tumefaciens is an important step in increasing the efficiency of recombinant protein production in plants. However, the limitation of this system that makes it economically non-competitive for industrial-scale applications is the Agrobacterium suspension production cost. In this study, the utilization of sugarcane molasses as an alternative low-cost source of carbon at a concentration of 8.7 g/L and nitrogen at a concentration of 2.4 g/L for Agrobacterium cultivation was investigated. Molasses pretreatment using sulfuric acid (SA) was applied before fermentation, and it resulted in a maximum specific growth rate of 0.232 ± 0.0063 h−1 in the A. tumefaciens EHA105 culture. The supplementation of antibiotics in the molasses-based medium was shown to be unnecessary for plasmid maintenance during fermentation in both Agrobacterium strains, which helped to reduce the production cost. We evaluated recombinant protein production using an Agrobacterium culture without antibiotic supplementation in the growth media by demonstrating green fluorescent protein expression in wild-type Nicotiana benthamiana leaves. In the evaluation of the culture medium cost, the molasses-based medium cost was 6.1 times lower than that of LB. Finally, this study demonstrated that the newly developed molasses-based medium for Agrobacterium fermentation is a feasible and effective medium for transient recombinant protein production in plant tissues.

Cordyceps militaris is an industrially important fungus, which is often used in Asia as traditional medicine. There has been a published genome-scale metabolic model (GSMM) of C. militaris useful for predicting its growth behaviors; however, lipid metabolism, which plays a vital role in cellular functions, remains incomplete in the GSMM of C. militaris. A comprehensive study on C. militaris was thus performed by enhancing GSMM through integrative analysis of metabolic footprint and transcriptome data. Through the enhanced GSMM of C. militaris (called iPC1469), it contained 1469 genes, 1904 metabolic reactions and 1229 metabolites. After model evaluation, in silico growth simulation results agreed well with the experimental data of the fungal growths on different carbon sources. Beyond the model-driven integrative data analysis, interestingly, we found key metabolic responses in alteration of lipid metabolism in C. militaris upon different carbon sources. The sphingoid bases (e.g., sphinganine, sphingosine, and phytosphingosine) and ceramide were statistically significant accumulated in the xylose culture when compared with other cultures; this study suggests that the sphingolipid biosynthetic capability in C. militaris was dependent on the carbon source assimilated for cell growth; this finding provides a comprehensive basis for the sphingolipid biosynthesis in C. militaris that can help to further redesign its metabolic control for medicinal and functional food applications.