The expanding field of alternative proteins represents a transformative approach to addressing global food security and sustainability challenges. Among these, fermentation-derived alternative proteins cultivated from microorganisms such as fungi, bacteria, and algae offer a promising avenue for sustainable protein production. This review explores the selection and utilization of raw materials to produce microbial proteins through fermentation processes. Critical raw materials include agricultural byproducts, industrial waste streams, and specifically designed feedstocks, which not only mitigate environmental footprint but also enhance the economic viability of production systems. The utilization of lignocellulosic biomass and molasses has demonstrated considerable promise, attributed to their abundant and renewable nature. The review underscored the necessity of exploring specific areas to enhance the viability of producing microbial protein from diverse raw materials. These areas include improving pre-treatment strategies to enhance substrate suitability for fermentation, optimizing fermentation processes for increased yield and reduced costs, and developing more resilient microorganisms capable of thriving on varied substrates. These strategies are crucial for advancing the production of alternative proteins through fermentation, in addition to raw material selection, which is vital in the scalability and sustainability of alternative protein production through fermentation, emphasizing the need for continued research and innovation in this field.
Interest in the technology for producing alternative proteins is rapidly increasing, driven by the need to find new ways to produce and consume protein for the global population. This technology involves growing different microorganisms and animal cells under controlled conditions to ensure their viability and efficient growth. The cultivation process takes place in different types of bioreactors, from traditional models to innovative new designs, each offering unique features and capabilities. The most commonly used bioreactors are stirred tank reactors, which are mechanically agitated, and airlift or bubble column bioreactors, which are pneumatically agitated. These bioreactors are often adapted or modified to optimize the production of cultured meat. Essential to the process are microcarriers or scaffolds that support cell adhesion and proliferation. Other bioreactor models, such as hollow fiber and packed bed bioreactors, are also being explored. The trend towards single-use technology is growing due to benefits like easier cleaning and sterilization, and reduced operation times, though it does raise concerns about plastic waste. This review not only describes various bioreactor models but also discusses instrumentation and control systems. It aims to present the main bioreactor models currently in use for cultivated meat production, detailing their features, advantages, disadvantages, and the technological challenges that need to be addressed.
Sludge generated at sewage treatment plants is of environmental concern due to the large production and the presence of a high amount of organic contaminants. Agricultural situations often include organic pollutants such as herbicides, insecticides, antibiotics, polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins, and polychlorinated dibenzofurans (PCDD/Fs). They are frequently transported into the environment as waste-water effluents due to their use in farm management practices and for human health plans.
The impact of these contaminants on plant health is also a critical concern, as they can affect plant growth, nutrient uptake, and overall ecosystem stability. This study examines advanced remediation technologies, including physical, chemical, and biological methods, for their effectiveness in addressing soil contamination from sludge. Emphasis is placed on the integration of advanced techniques like phytoremediation, bioremediation, and soil washing, alongside emerging technologies such as artificial intelligence (AI) and machine learning (ML) for enhanced site characterization, contaminant detection, and optimization of remediation strategies. By enhancing the accuracy, efficiency, and sustainability of remediation efforts, these technologies hold the potential to significantly mitigate the environmental and health risks associated with organic contaminants in soil and their impacts on plants.
Enzymes play a pivotal role in meat processing and maturation, starting with endogenous enzymes that catalyse a partial proteolysis in natural meat that enhances texture and flavor, to exogenous enzymes that may be incorporated for additional processing. Therefore, a question is raised whether endogenous and exogenous proteases could be used to improve the quality of meat products originated from in vitro cell-based technology. This manuscript aims at presenting the main applications of enzymes in meat processing, starting with conventional meat products and expanding to reported and potential applications in cultured meat technologies. Enzymes are applied since the early stages of cell isolation, multiplication and detaching from supports. The use of endogenous enzymes, such as cathepsins and calpains, has been proposed to improve the palatability of cultivated meat, and the application of transglutaminases has already been reported in scientific papers and patent documents, with the aim of promoting crosslinking reactions among proteins. This feature can be used to modulate the texture of cultivated meat products. Also, their use has been directed to the manufacture of scaffolds and microcarriers for animal cell cultivation. As concluded from the scientific and technological literature survey, it is evident that a substantial area of study remains unexplored regarding the utilization of enzymes in cultivated meat products.
Food waste and by-products are generated throughout the food processing and storage chain. In a world facing climate collapse and limited space for expanding cultivable land needed to feed a growing global population, utilizing food from sustainable production chains has become a significant challenge. Additionally, there is a worldwide trend towards consuming natural and healthy foods that are as free from chemical compounds as possible during production, processing, and preparation. Gradually, eating habits have adapted to these new trends, and new foods are being introduced into diets. In this context, research into sustainable practices has emerged worldwide, promoting the increased consumption of plant-based foods. The central idea of this article is connected to global concerns regarding food sources, minimizing waste, and innovatively using every ingredient. Fermentation, a traditional and natural food preservation technique, can be a vital tool for enhancing flavours and textures while increasing nutritional value through the action of specific enzymes. This article aims to highlight the main challenges of using food processing by-products in human nutrition and explore possible strategies to improve their quality through the enzymatic action of microorganisms.
Whey protein concentrate (WPC) via single or sequential enzymatic hydrolysis steps using either only Alcalase® 2.4 L (WPH-A) or Alcalase® 2.4 L followed by Flavourzyme® 500 L (WPH-AF) was investigated. Different bioactive properties of particular interest in the obtained hydrolysates were evaluated. Calcium-chelating capacity (Ca-CC) of the hydrolysates was optimized using response surface methodology to adjust the experimental conditions used for producing the calcium nano-compounds. Chelates with higher Ca-CC were obtained using the WPH-AF compared to WPH-A. Additionally, antihypertensive and antioxidant activities of both hydrolysates showed significant improvements when compared to WPC, and these capabilities remained stable after undergoing different heat treatments. Finally, both hydrolysates presented a cytoprotective effect in two cell lines, suggesting that these products may have positive effects against diseases associated with reactive oxygen species. The results obtained indicate that the WPHs produced herein could be used in the development of food formulations with potential health benefits.
The long fermentation period constitutes a crucial process for enhancing the quality of base liquor in the production of Chinese strong-flavor Baijiu. In the present study, a comprehensive metaproteomic analysis of fermented grains in the long fermentation period led to the identification of 44 plant proteins and 1460 microbial proteins. Following 12 days of fermentation, bacterial proteins exhibited a rapid surge, constituting the predominant components of fermented grains proteins, ranging from 64.64 to 81.28%. The differential proteins were predominantly associated with carbohydrate metabolism, energy metabolism, and amino acid metabolism. Among these, days 18 to 220 were dominated by differential protein in the protein hydrolase system and amino acid metabolic of the bacteria. Pathway analysis delineated the potential metabolic pathways through which cysteine, tyrosine, proline, isoleucine, among others, could be metabolized to yield pyruvic acid, acetyl-CoA, ultimately leading to the production of acetic acid, butyric acid, and hexanoic acid. Furthermore, phenylalanine underwent metabolism, resulting in the production of phenylacetaldehyde, phenylacetic acid, and phenylacetic acid ethyl ester. With the extension of fermentation time, lactic acid underwent conversion to pyruvic acid, contributing to the generation of other acids, while aldehydes were converted to alcohols and acids.
The study focused on the regulatory role of Carthamus tinctorius Dof in the safflower seed oil biosynthetic pathway. Through bioinformatics analysis, yeast expression system validation, and transgenic Arabidopsis thaliana experiments, it was found that the expression of the safflower family of transcription factors was significantly increased at 25 post-flowering days of safflower seed development, which coincided with the peak of fatty acid accumulation. Stearic acid content was reduced by 20.33% and oleic acid content was increased by 27.54% in transgenic yeast, and C20:0 and C22:0 long-chain saturated fatty acids were detected. Arabidopsis thaliana seeds overexpressing the safflower Dof transcription factor gene had significantly higher fatty acid composition than the wild type, while mutant seeds had lower fatty acid composition than the wild type. GC–MS analysis indicated that the safflower Dof transcription factor gene variant had limited effect on the overall composition of fatty acids. The results provide molecular targets for improving the quality of safflower seed oil and help to reveal the mechanism of Carthamus tinctorius Dof in the regulation of lipid biosynthesis, which is of great significance for improving the lipid content and composition of oilseed crops.
As a saccharifying and fermenting starter, jiuyao is indispensable for huangjiu brewing by providing abundant microorganisms and hydrolytic enzymes. However, the current production of jiuyao still relies on the preceding year’s jiuyao inoculation and natural fermentation. Due to the unpredictable and unstable assembly of core microbial community, the quality of jiuyao fluctuates across different batches, thus the quality of huangjiu suffers subsequently. In this study, we took jiuyao as a case study to identifying and reconstructing the core microbiota. Five species, Pediococcus pentosaceus, Saccharomycopsis fibuligera, Saccharomyces cerevisiae, Mucor indicus, and Rhizopus microsporus, were comprehensively identified as the core microbiota. A synthetic microbiota was constructed through inoculating the core microbiota to artificial jiuyao, and further supplemented by the spontaneous inoculation of geography-dependent microorganisms (Weissella cibaria and Rhizopus arrhizus) from original factory environment during open fermentation. The artificial jiuyao ultimately exhibited comparable microbial community and physicochemical indexes with traditional jiuyao. Specifically, their gelatinized-starch-hydrolyzing glucoamylase activities were 343.28±32.27 and 340.59±39.80 U/g respectively. Furthermore, huangjiu brewed with artificial and traditional jiuyao showed similar physicochemical and flavor profiles, with the ester content of the former being 5.32% higher and the content of higher alcohols being 9.64% lower compared to the latter. These results suggested that the rational synthetic core microbiota could substitute preceding year’s jiuyao and facilitate production to be controllable and tractable. Combined with a specific production environment, it could effectively reproduce the function of jiuyao and the terroir flavor of huangjiu, providing a scientific guidance for similar fermentation control and optimization.
The microbiome in the gastrointestinal tract (GIT) of fish influences host health in both beneficial and detrimental ways and understanding the gut microbiota requires 16S amplicon sequencing. Although, Cirrhinus reba (Hamilton, 1822) has achieved widespread recognition for its nutritional and commercial value, the carp are currently confronting production limits. Probiotic-based carp captive breeding may be an effective method for enhancing the production and health condition of the carp and it requires conventional culture-based analysis. Hence, a comprehensive study was performed on the intestinal microbiota of C. reba by both 16S amplicon sequencing and culture-based approaches. At the phylum level, Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, and Fusobacteria were dominating the GIT of the fish. Importantly, Gram-negative pathogenic genera like Pseudomonas, Serratia, Aeromonas, and Unclassified Clostridiales are found to be most abundant. Only Bacillus and Lactobacillus are found as the Gram positive bacterial genera among the top ten dominating bacteria in the GIT of the fish. The total load of culturable bacteria in the gut of the carp was 1.12±0.26×108 cfu/g comprising twenty-four different types of colonies. The Gram negative bacterial load in the GIT of the carp is 6.9×107 cfu/g. Only 37.5% (4.2×107 cfu/g) of the isolates were Gram-positive and rod-shaped. Among them, one intestinal isolate (PKS9A) had shown significant antagonism against four common fish pathogens. The isolate was positive for the production of extracellular digestive enzymes like Protease, Amylase, Cellulase, Xylanase, Lipase, Phytase. The isolate PKS9A exhibited sufficient probiotic properties like bile salt hydrolase activity, cell surface hydrophobicity, antibiotic sensitivity, and is non-hemolytic in nature. 16S rDNA sequencing and phylogeny analysis identified the isolate as Bacillus paramycoides PKS9A (Accession no.-OR003914). Hence, the isolate may be utilized as a potential autochthonous probiotic for the cultivation of C. reba to enhance the nutritional and health status of the carp and to protect it from further deterioration.
Recombinant HCGβ-LTB expressed in the yeast, holds a significant promise as a contraceptive vaccine for women. It induces bio-effective anti-hCG antibodies, which can potentially prevent pregnancy by neutralizing the hCG hormone crucial for establishment and maintenance of pregnancy. The vaccine produced in shake-flask culture and administered along with adjuvant Mycobacterium indicus pranii (MIP) induces high anti-hCG titres in mice of different genetic strains. This study aimed to produce the vaccine in high cell density culture in a bioreactor and assess its consistency and efficacy compared to shake-flask production. A fed-batch culture increased the cell biomass nearly 6.5 folds higher than the shake-flask culture. Increase in specific-activity by a factor of 1.7 resulted in almost 11-fold higher volumetric activity. Characterization of the purified protein and immunogenicity studies conducted in mice demonstrated that it was comparable to protein made in shake-flask. This study shows that hCGβ-LTB vaccine can be produced cost-effectively with consistent characteristics in high cell density cultures of Pichia pastoris on a large-scale for eventual pre-clinical/clinical studies.
The current study deals with cloning and expressing a maltogenic α-amylase gene from thermophilic Bacillus stercoris YSP18 (AmyYSP) in Escherichia coli BL21 (DE3). AmyYSP belongs to the GH13_20 subfamily of Glycoside Hydrolases and entails five conserved regions found in maltogenic α-amylases. As a monomer of 67 kDa, AmyYSP exhibits maximal activity at 80°C, pH 5.0 and retains>75% residual activity at 70–100°C and pH 5.0–8.0. The kinetic and thermodynamic studies displayed that it has a high affinity for soluble starch (Km = 1.54±0.236 mgmL−1), exhibits a longer half-life (38.5 h at 80°C and 8.88 h at 100°C), and a higher EaD value of 3824±1.03 kJ mol−1. It was characterised as a Ca2+-independent α-amylase, resistant to various denaturing additives. It hydrolyses soluble starch and raw corn starch efficiently, liberating glucose, maltose, maltotriose and maltotetraose as the main products. The thermostable and acid-stable, maltooligosaccharide forming AmyYSP is a versatile enzyme with prerequisites for successful application in starch-saccharification industries.
This study utilized natural potato starch and optimized process conditions to establish an efficient cyclodextrin (CA) production system through the combination of a mutant 4-α-glucosyltransferase (TaAM-Y54G) from Thermus aquaticus and pullulanase from Bacillus deramificans. Under optimal conditions (1% potato starch concentration, 65 °C for 12 h), CA conversion rate reached 43.2%, a new record for natural industrializable substrates. To address solubility issues, TaAM-Y54G was used for liquefaction at 70 °C for 10 min, stabilizing conversion rates at 29.6% and 22.3% under 5% and 10% substrate concentrations, respectively. Furthermore, this study optimized the fermentation process of the mutant recombinant strain, achieving the highest enzyme activity of TaAM-Y54G at 2525.9 U/mL, significantly improving the fermentation level to the highest reported level. These innovations significantly increased CA yield, laying a solid foundation for industrial production and showing broad application potential.
Selecting the appropriate algal species is essential to maximize lipid extraction from microalgae. Chlorococcum sp. is a unicellular organism found in both aquatic and terrestrial habitats. Although the influence of nitrogen on lipid metabolism is well established across various microalgae, its particular effects on Chlorococcum sp. provide novel insights into this relatively underexplored species. Examining how nitrogen affects lipid accumulation in Chlorococcum sp. could reveal new strategies to enhance its application in biodiesel productions. This study aimed to evaluate the lipid content of Chlorococcum novae-angliae and compare the lipid and fatty acid yields between cultures grown in N-supplied and N-starvation culture media. The results show that Chlorococcum sp. cultivated in N-supplied culture medium reached the highest cell count of 2.16±8.18×10−8 cells/mL, with a specific growth rate (µ) of 0.55 d−1, whereas a cell count of 1.60±6.63×10−8 cells/mL was found for the N-starvation culture medium. On the other hand, the highest lipid yield, recorded as 0.098±0.012 g lipid/g wet biomass, mainly consisting of tridecanoic and palmitic acids (77%), was obtained from the N-starvation culture medium. Tridecanoic acid (C13:0) was detected for the first time in C. novae angliae. For the N-supplied culture medium, the lipid yield was 0.082±0.010 g lipid/g wet biomass. Therefore, in cases where maximizing lipid yield is crucial, such as for biofuel production, nitrogen starvation might be a more effective approach, even though it may result in lower overall biomass productivity. However, for applications that prioritize higher biomass, such as animal feed, ensuring sufficient nitrogen levels could be more beneficial.
6′-sialyllactose (6′-SL) is an important component of human milk oligosaccharides (HMOs) and has numerous infant health benefits. The construction of efficient and food-safe microbial cell factories to produce 6′-SL has attracted increasing attention. In this study, a Bacillus subtilis strain was metabolically engineered for 6′-SL production. First, a de novo synthesis pathway for 6′-SL was constructed by heterologous expression of neuC, neuB, neuA, and pst6, enabling 6′-SL synthesis at a titer of 135.17 mg/L. Subsequently, bioinformatics-guided enzyme modification and promoter substitution strategies were used to fine-tune the pathway strength. Moreover, inhibition of competing pathways and copy number optimization of synthetic modules were used to increase the precursor concentration, raising 6′-SL titer to 621.8 mg/L. Furthermore, a strategy to overcome carbon catabolite repression (CCR) was developed for B. subtilis to improve lactose utilization and increase 6′-SL titers, reaching 3.55 g/L in shake flasks and 15.0 g/L in 3-L fermenters. This study established a foundation for efficient 6′-SL production.
The low-expression level of lactoferrin (LF) in the production process poses a significant challenge. This study aimed to efficiently express bovine lactoferrin (BLF) using Pichia pastoris GS115 as the expression host and PIC9K as the recombinant vector. Optimization strategies included codon usage, promoter selection, and fermentation conditions. The blf gene was optimized for P. pastoris GS115 bias, resulting in the construction of the recombinant vector pPIC9K-UBLF1-3 controlled by the AOX1 promoter. SDS-PAGE analysis revealed soluble and efficient expression of ublf3 in P. pastoris GS115, with a molecular mass of approximately 76 kDa. The transformant P. pastoris GS115/pGAP9K-UBLF3-4 resistant at 4 mg·mL−1 G418, exhibited a ublf3 gene copy number of 5.88 through high-copy screening. Optimal expression conditions of recombinant UBLF were determined as 24℃, pH 5.0 and 220 r·min−1 through fermentation condition optimization. Under these conditions, recombinant UBLF production reached 40.62 mg·L−1. The yield of recombinant UBLF was reached 824.93 mg·L−1 through high-density fermentation. Antibacterial assay demonstrated the efficacy of recombinant UBLF against Escherichia coli JM109 and Staphylococcus aureus CGMCC 1.282. This study successfully achieved the efficient heterologous expression of recombinant UBLF in P. pastoris GS115, providing valuable insight for industrial production and the potential development of natural antibacterial agents.
β-Alanine is the only naturally occurring β-type amino acid, with various applications in the pharmaceutical, food, and chemical industries. Given the growing market demand, the study of β-alanine production is important. This study utilized a modified lysine-producing strain as a chassis cell line to further promote β-alanine synthesis through metabolic engineering. In order to reduce the consumption of oxaloacetate, the gene pck was deleted. A promoter mutation library was constructed to screen the original promoter of the stronger promoter replacement gene pyc to enhance the oxaloacetate synthesis pathway and further increase the intracellular supply of oxaloacetate. Next, the gene poxB was deleted, and pyruvate accumulation further promoted β-alanine synthesis. Then, the aspartate kinase-coding gene lysC was weakened by predicting the RBS sequence, thus reducing the synthesis of lysine by-products and improving β-alanine synthesis. Ultimately, the carbon flux in the β-alanine biosynthetic pathways was increased by overexpressing aspartate-α-decarboxylase, aspartate ammonia-lyase, and aspartate aminotransferase using the strong promoter Ptrc. The resulting strain QBA9 was cultured in a 5-L fermenter by fed-batch to produce 70.8 g/L of β-alanine with a productivity of 0.98 g/L/h. These modification strategies demonstrate the potential for efficient β-alanine production by the lysine-producing strain and provide an innovative idea for the developing β-alanine-producing strains.
The production of L-alanine was enhanced in Corynebacterium glutamicum ATCC13869 through metabolic engineering of the biosynthesis pathways of L-alanine and fatty acids. Strains ΔfasB, ΔfasBR, ΔfasBΔpks13 and ΔfasBRΔpks13 were constructed and exhibited increased L-alanine yields up to 17.29 g/L. Different from ΔfasB mutant constructed from C. glutamicum ATCC13032 in which L-glutamate production accumulated, the muatnt ΔfasB constructed from C. glutamicum ATCC13869 in this study significantly produce L-alanine without L-glutamate accumulation. Transcriptional level analysis revealed that the knockout of fasB upregulated the expression levels of the genes related to L-alanine synthesis but downregulated those associated with fatty acid synthesis, confirming the redirection of metabolic flux from fatty acid synthesis to L-alanine synthesis in these strains. L-alanine productions were further enhanced in strains ΔfasB and ΔfasBR through the combinatorial expression of heterologous genes Bacillus subtilis alaD encoding alanine dehydrogenase and Escherichia coli alaE encoding alanine export protein, and the yields reached 55.21 g/L and 54.95 g/L, respectively. Finally, 69.9 g/L L-alanine was obtained in ΔfasB/pJYW-5-alaDE after 60 h of fermentation by supplementing glucose. Our data indicate that disrupting the fatty acid biosynthesis could redirect metabolic flux towards L-alanine biosynthesis. These results provide a new strategy for increasing the production of L-alanine in C. glutamicum.
Corynebacterium glutamicum is widely used in the production of amino acids. C. glutamicum possesses seven sigma factors, among which SigD is responsible for the transcription of genes involved in the synthesis of mycolic acid (MA) and its derivatives, the unique cell envelope of C. glutamicum. To understand the influence of MA synthesis on amino acid production and membrane phenotype of C. glutamicum, the expression of sigD gene and some mycolyltransferase genes, i.e., cmt1, cop1 and cmt2, were regulated by several growth-regulated promoters in this study. Except for 2 mutant strains of Pcg3096-sigD and Pcg1633-cop1, the growth and 4-hydroxyisoleucine (4-HIL) titer of most modified strains did not change significantly. But the 4-HIL titer of PodhI-sigD strain increased by 20.73% (142.45±3.69 mM) compared to that of control strain (117.99±0.34 mM). After it was cultivated in bioreactor, 4-HIL titer reached 372.56 mM. This may be caused by the increase of MA content, and 17% decrease of cell hydrophobicity and 12% increase of membrane permeability were observed at the exponential phase. In conclusion, we proved that rearrangements in regulation of sigD expression contributed to the improved fermentation performance of C. glutamicum and promoted 4-HIL production.
This study described that a low-producing mutagenic strain was transformed to a l-lysine high-producing recombinant strain by optimizing the l-lysine metabolic pathway of Corynebacterium glutamicum. The nucleotide sequence results revealed that the lysC of mutant strain CgK37 mutated at 279th codon. Based on this site, site-directed saturation mutation was performed to screen for the mutant with better effect in relieving aspartate kinase feedback inhibition. Then, the supply of oxaloacetate and nicotinamide adenine dinucleotide phosphate was increased via knockout and overexpression of related genes. In order to solve the problem of low utilization efficiency of culture medium, fructokinase gene gmuE was heterologous expressed in CgK37, which could directly use intracellular fructose to improve the growth rate. In addition, the synthesis of partial by-products was weakened at the gene transcription level to avoid carbon excessively flowing into the branch metabolism. Finally, a large-scale fermentation experiment was conducted in 5 L jar fermenter. The l-lysine yield of CgK37-11 was 196.58±1.68 g/L, which was 83.24% original higher than CgK37, and the productivity reached 2.46 g/L/h.
Xylonic acid is a versatile and valuable chemical with a range of industrial and potential health applications. The high demand for renewable energy and sustainable products has increased interest in xylonic acid as a starting material for the production of variety of high value chemicals and biofuels. The production of xylonic acid from biomass have an added advantage that it provides an opportunity to utilize bio-waste materials that would otherwise be discarded, reducing environmental hazards and reducing waste disposal costs. The aim of this research was to establish a bioprocess for producing xylonic acid from acid pre-treated sawdust liquor via fermentation, utilizing metabolically engineered strain of Corynebacterium glutamicum ATCC 31831. A maximum titre of 48.5±0.2 g/L xylonic acid from 60 g/L xylose derived from biomass was obtained with a yield of 0.89 g/g xylose which corresponds to a conversion of 62%. 30.8±0.4 g/L of xylonic acid crystals of 95.5% purity were successfully recovered from 1L sawdust APL (Acid Pre-treated Liquor) via solvent precipitation and crystallization. Purified xylonic acid crystals were checked for its antibacterial effect on Salmonella enterica MTCC 3224, Escherichia coli MTCC 443 and Staphylococcus aureus MTCC 96.
Bacillus thuringiensis, a safe bacterium widely used in agriculture for the biocontrol of pests, has great potential for protein production. The linear plasmid expression system, bacterial orthogonal DNA replication system constructed based on B. thuringiensis prophage GIL16, can achieve stable and high levels of gene expression in the absence of external selection pressure, facilitating development of B. thuringiensis chassis cells. However, the regulatory elements of gene expression and protein secretion suitable for the B. thuringiensis expression system are still lacking. Therefore, the development and optimization of different genetic tools are required. We constructed a promoter library containing 107 different-strength promoters (covering persistently high/intermediate/low level) by transcriptomic analysis of the cell at different growth stages and a signal peptide library (59 signal peptides from Bacillus subtilis and four endogenous signal peptides from B. thuringiensis) to enrich the genetic toolbox using alpha-lactalbumin (α-LA) as the characterization product. Then, a high-throughput microfluidic screening platform based on BacORep and self-assembled split fluorescent protein was developed to further optimize expression elements, resulting in an improved α-LA-producing B. thuringiensis. Finally, the maximum copy number of linear plasmids was 9.3 times higher than that of the original. The titer of α-LA reached 107.7 mg/L in a 3 L bioreactor, which was comparable to the highest yield reported in Komagataella phaffii. We substantially expanded the synthetic biology toolbox for linear plasmid expression systems and provided a strategy for creating efficient prokaryotic expression system.
Microalgae and cyanobacteria are photosynthetic microorganisms that inhabit freshwater and marine ecosystems. Bioactive substances (metabolites such as astaxanthin, chlorophyll-a, and phycobiliproteins) obtained from microalgae and cyanobacteria are used in a multitude of fields. Phycobiliproteins are photosynthetic antenna pigments that are found in cyanobacteria, red algae, and cryptophytes. This study aimed to determine the optimal parameters for phycobiliprotein extraction from lyophilized cells obtained from a triple algal co-culture. These parameters included the biomass: solvent ratio, CaCI2 concentration, agitation speed, and extraction time. In all optimization processes, phycocyanin is observed to be the most dominant, while phycoerythrin has the lowest amount. It is demonstrated that all phycobiliprotein efficiencies increase after each optimization process. The highest yield of 12.51±0.23 mg phycobiliprotein/g freeze-dried weight was obtained using a 1:100 (v: v) biomass: solvent ratio with 2% CaCl₂ at 100 rpm for 1 h. The significance of carefully controlling extraction parameters to maximize the efficiency of PBP extraction from triple algal co-culture is highlighted by these results. Employing a combination of extraction methods could potentially improve both the yield and purity of phycobiliproteins obtained from a triple algal co-culture. Future research should focus on developing and refining scaling-up techniques to enhance and optimize the extraction process of phycobiliproteins for industrial use.
The combined inoculation of yeast and lactic acid bacteria (LAB) is a promising approach to enhance microbial metabolism and the quality of fermented food products. In this study, we investigated the effects of simultaneous and sequential inoculation of LAB and yeast on coffee fermentation. Fermentations were conducted using single and combined protocols with P. fermentans YC5.2 and P. pentosaceus LPBF07. A temporal analysis utilizing Illumina high-throughput rRNA Gene Sequencing revealed a synergistic interaction between the two microbial groups. This positive synergy led to increased consumption of coffee pulp sugar and the production of metabolites, surpassing the results observed in single cultures and the spontaneous process. Furthermore, the combined inoculation processes demonstrated a more significant role in suppressing wild microbiota compared to single cultures. Notably, the sequential process emerged as particularly effective in promoting a more intricate aroma profile and elevated sensorial score, attributed to the formation of distinctive compounds such as benzeneacetaldehyde, 1-hexanol, 2-heptanol, benzyl alcohol and phenylethyl alcohol. These results suggest that implementing a sequential inoculation process could enable coffee farmers to standardize on-farm processing and produce high-value-added products.
Trehalose is a widely used and safe natural disaccharide. Maltooligosyl trehalose hydrolase(MTHase) is one of the key enzymes for trehalose preparation by double enzyme method using starch or dextrin as substrate. In industrial production, the thermalstability of MTHase is of great significance. We first heterogeneously expressed MTHase from Arthrobacter in E.coli strains BL21 (DE3). Based on the overall stability of the protein after virtual saturation mutation predicted by FoldX and the evolutionary information from PSSM, 15 mutations were selected and combined. Finally, the combinatorial mutant G589P/A57P was obtained. At 60 ℃, the t1/2 of G589P/A57P and wild type are 37 min and 19 min, respectively, which is 1.9 times higher than that of wild type. The enzyme kinetic parameters of G589P/A57P were analyzed. The KM and kcat are 4.82 mM and 1136 s−1, respectively, and the results were close to the wild type, indicating that the mutation did not reduce the catalytic efficiency of the enzyme. The molecular dynamics simulation results show that the rigidity and thermal stability of G589P/A57P protein increase in the range of residues 50–100 and 400–500, which may be due to the proline effect caused by the introduction of proline.
Buildings contribute around 37% to global carbon emissions, prompting a growing interest in innovative carbon capture technologies. Among these, the integration of microalgae-based photosynthesis into building facades has emerged as a promising solution. This approach offers multiple benefits, including carbon sequestration, reduced energy consumption, dynamic shading, and improved thermal regulation. This paper investigates the impact of integrating photobioreactor (PBR) facade elements, specifically on the south-facing facade of an office building in a temperate continental climate. The study evaluates the system’s effects on indoor thermal and visual comfort, energy production, and carbon dioxide (CO2) sequestration for three distinct PBR facade alternatives and compares them with a commercial curtain wall. The continuous PBR system varies in performance depending on production intensity, necessitating an initial optimization for thermal and visual comfort alongside energy use. Simulations were conducted using Rhinoceros/Grasshopper plug-ins, with optimization performed via the Octopus tool. The results, focusing on the Chlorella vulgaris algae strain, demonstrate that all facade configurations achieve a daylight performance exceeding 50% and meet desired thermal comfort levels. Although the energy generated by the PBR facade does not fully offset the building’s energy consumption, annual CO2 sequestration ranges from 84.87 kg to 770.13 kg. This study concludes that microalgae facades offer a viable strategy for enhancing a building’s energy performance and reducing CO2 emissions, without compromising occupant comfort. Additionally, the findings provide valuable insights for designers, researchers, investors and stakeholders and provides a payback period of these systems (16–24 years) for commercialization in the building industry.
Cell culture media is a significant contributor to the high cost of bioprocesses. This study explored the potential of algae to re-condition spent cell culture medium, which may reduce the costs of pharmaceutical and lab-grown-meat manufacturing. Chlorella sorokiniana, a thermally resistant microalgal species, exhibited heterotrophic and mixotrophic growth in mammalian cell culture growth media (GM). Spent animal cell media, generated by culturing quail myoblast cells (QM7s) for 4 or 8d (4D-SGM and 8D-SGM, respectively), was harvested for algal culture. Increased algal growth was observed in 4D-SGM, when compared to fresh media after 3d (optical density of 1.39±0.22, 0.47±0.17, p≤0.05). Within 72 h, ammonia and glucose were eliminated from 4D-SGM with algal treatment. After treating 4D-SGM with algae, the treated media was reintroduced into QM7 cell cultures. No cytotoxic effects were observed on QM7 cells grown in algal-treated growth media and QM7 cells exhibited better metabolic activity in algal-treated spent medium than in untreated spent medium (80.85±12.02% and 44.57±10.82% activity of fresh media group, respectively, p≤0.05). These results suggest that C. sorokiniana can be grown in spent media at 37 °C, sequester ammonia, and potentially extend the lifespan of media, thereby enabling more affordable bioprocesses.
Growing interest in the abatement of greenhouse gases has spurred a surge in research within the field of methanotrophy in recent decades. Certain methanotrophic organisms exhibit the unique ability for the production of biodegradable polymers known as polyhydroxyalkanoates (PHAs) using methane as their solitary carbon source. In this study, we harnessed the capability of Methylocystis suflitae, a Type II methanotroph, for the production of PHA from methane. The genome analysis unveiled the presence of four paralogs of PHA synthase gene in Methylocystis suflitae. Subsequently, we elucidated the catalytic sites of each PHA synthase using protein modeling and molecular docking. Both hydroxybutyrate and hydroxydodecanoate demonstrated the highest docking energies among all the tested substrates, recording at − 7.5, and − 7.8 kcal/mol, respectively. The capability of Methylocystis suflitae to synthesize polyhydroxybutyrate (PHB) was evaluated by analyzing the FTIR spectrum, revealing the characteristic carbonyl (C=O) peak at 1723 cm−1. The study included the optimization of the substrate-to-electron acceptor ratio to optimize PHA productivity. Notably, the organism exhibited a productivity value of 11.90±1.34 mg PHA/L/hr. This higher productivity holds significant promise for industrial PHA production, particularly in scenarios where achieving sufficiently high dissolved methane concentrations in industrial fermenters is inherently challenging, potentially enabling more efficient PHA production. Additionally, we determined the melting temperature for PHB produced by Methylocystis suflitae which closely aligns with the standards of commercial-grade PHB, at around 188 °C.
To achieve high production of arachidonic acid (ARA) through industrial fermentation of Mortierella alpina, the correlation between metal ions, mycelium pellet morphology and ARA production was investigated. By adding different metal ions to the culture medium, observing the morphological differences of mycelium pellet, total oil production and ARA content and established a correlation between them. The results showed that adding sodium ions increase the density of mycelium pellet, while reducing the diameter of mycelium pellet, resulting in a 41.59% increase in ARA production. Potassium ions, magnesium ions, calcium ions and iron ions slightly reduce the diameter of fungal hyphae and increase ARA production by 7.47%, 17.81%, 36.05% and 9.37%, respectively. At the same time, calcium ions promoted the branching growth of mycelium, which solve the problem of excessive free mycelium in fermentation broth within a certain concentration range. Zinc ions had a negative impact on bacterial growth, leading to the formation of mycelium clumps in the fermentation broth. Manganese ions and ferrous ions could transform the morphology of fungal mycelium into a smooth spherical surface, which was not conducive to the growth of M. alpina and the accumulation of oil. The comprehensive results showed that the optimal fermentation mycelium form for M. alpina was a dense central and loosely packed mycelium pellet. The yield of ARA was directly proportional to the density of mycelium pellet and inversely proportional to the diameter of mycelium pellet.
Bacteroides thetaiotaomicron colonizes the human gastrointestinal tract and establishes a symbiotic relationship with the host, contributing to reducing intestinal inflammation and enhancing resistance against foreign pathogens. Recent reports have revealed that diverse lipid species such as glycerophospholipids, sphingolipids, and N-acyl amines exist in B. thetaiotaomicron and play essential roles in the immune process. In this research, total lipids obtained from B. thetaiotaomicron were purified via thin-layer chromatography, and the species and molecular structures of visible lipids in different hydrophobic regions were qualitatively characterized by high-performance liquid chromatography-mass spectrometry. The results indicated that seven lipid species were primarily displayed on the plate, including phosphatidylethanolamine, ethanolamine phosphoryl dihydroceramide, inositol phosphoryl dihydroceramide, glycyl-serine phosphoryl dihydroceramide, phosphatidylglycerol, cardiolipin, and glycyl-serine phosphoryl diacylglycerol. The phosphatidylethanolamine, ethanolamine phosphoryl dihydroceramide, and inositol phosphoryl dihydroceramide species corresponding to ion peaks at m/z 676.48, 691.53, and 796.53 exhibited significantly high abundance compared to other species, suggesting their prevalent presence in total lipids. The molecular structures of phosphatidylethanolamine and ethanolamine phosphoryl dihydroceramide were derived from the modification of diacylglycerol and dihydroceramide with phosphoethanolamine, while the structure of inositol phosphoryl dihydroceramide was derived from the modification of dihydroceramide with phosphoinositol. The phosphatidylglycerol and cardiolipin species corresponding to m/z 721.51 and 1323.94 have been detected in the membrane lipids of B. thetaiotaomicron, although they were not mentioned in previous studies. These findings are important for understanding the molecular mechanisms of B. thetaiotaomicron colonization in mammalian gut.
L-serine is utilized in various applications across the pharmaceutical and food industry. Corynebacterium glutamicum, a non-pathogenic strain, is extensively used in amino acid production. However, the current titer and productivity of L-serine through direct fermentation are insufficient to meet the demands of industrial production. This shortfall arises from the strain’s inadequacy sucrose utilization, which affects both L-serine productivity and sucrose consumption rate. To solve the problem, this research conducted three strategies to increase the carbon flow from sucrose to L-serine. Initially, ALE was performed using a stress of 300 g/L sucrose based on A36-pDSer, and a biosensor-assisted high-throughput screening platform was utilized to identify the mutants with higher intracellular L-serine concentration. The strain A36-mut achieved 39.0 g/L L-serine titer, marking a notable 25.3% improvement over the parent strain A36 (31.1 g/L). Subsequently, the overexpression of the L-serine exporter serE along with its transcription factor serR in strain A36-mut led to an improvement in the L-serine production, reaching 44.8 g/L. Finally, by optimizing the fed-batch fermentation process, the L-serine titer and productivity were improved to 53.7 g/L and 0.50 g/L/h, respectively. This research presented the highest L-serine titer from sucrose in C. glutamicum to date, offering the possibility for the industrialization production of L-serine by fermentation.
The aim of this work was to evaluate the use of stover from brown midrib (bmr) grain sorghum to produce polyhydroxyalkanoates (PHAs). These mutants produce lignin with novel compositions and/ or reduced lignin content that was hypothesized to result in higher PHA production than when using a wild-type sorghum stover. Five sorghum stover samples [wild-type (control) and four bmr mutants [bmr2, bmr6, and bmr12, and a double bmr mutant: bmr6 and bmr12 (stacked)] were evaluated as substrates for poly(3-hydroxybutyrate) (P(3HB)) production. When the main sugars in non-detoxified alkali-pretreated sorghum stover hydrolysates (SSHs) were metabolized by Paraburkholderia sacchari for P(3HB) production, the SSHs from bmr plants were demonstrated to be promising feedstocks with a maximum P(3HB) concentration of 7.2 g/L from the bmr6 SSH, while a maximum P(3HB) concentration of 4.2 g/L P(3HB) was produced using wild-type SSH. Moreover, the highest maximum Y[P(3HB)/sugars] were achieved using stacked SSH and bmr12 SSH (0.44 g/g and 0.42 g/g, respectively). Furthermore, Cuprivadus necator was utilized to evaluate P(3HB) synthesis from the alkaline pretreated liquor (APL) remaining after sorghum stover pretreatment and an oxidative enzyme-mediator-surfactant system (OEMS) was used to improve P(3HB) production from the APLs. A 28-fold increase in PHB production from the bmr2-APL with OEMS compared to the fermentation of the same APL with no OEMS was observed. This is the first published report to use grain sorghum stover to produce polyhydroxyalkanoates (PHAs). Also, a novel combination of PHA production from both sugars and lignin is reported.