Microbial Biotechnology最新文献

L-(+)-tartaric acid (L-TA) is a crucial hydroxy carboxylic chelator with extensive applications in the food and pharmaceutical industries. The synthesis of L-TA from renewable biomass presents a promising approach to mitigating environmental impact and advancing green energy initiatives. Previous studies revealed that a mutant transketolase (TKTA_M) could catalyse the production of tartaric semialdehyde, a precursor to L-TA. This study focuses on the development of a Gluconobacter oxydans cell factory for tartaric semialdehyde production, employing a combination of metabolic engineering and a modular strategy. The genetically modified G. oxydans T strain exhibited robust expression of the tktA_M gene. The optimal pH and temperature for this strain were determined to be 6.0°C and 30°C, respectively. Under these conditions, the strain produced 32.21 ± 0.74 g/L of tartaric semialdehyde from glucose. Implementation of a “Push-Pull” strategy enhanced tartaric semialdehyde production, resulting in a 23.85% increase in the G. oxydans T02 cell growth. In CSLP medium with 100 g/L glucose, the fermentation process yielded 48.88 ± 2.16 g/L of tartaric semialdehyde and 7.72 ± 1.56 g/L of residual 5-KGA after 48 h. This resulted in a tartaric semialdehyde productivity rate of 1.018 g/L·h, representing an 87.82% improvement over flask fermentation. This study demonstrates a straightforward and efficient microbial process for the oxidation of glucose to tartaric semialdehyde, indicating its potential for industrial-scale production and facilitating the synthesis of L-TA from renewable resources.

Re-utilising brewers' spent grain (BSG) through LAB fermentation can enable its broad use in the food industry, enhancing its nutritional and functional properties and offering a clear example of a sustainable approach in the valorisation of food side streams. Despite extensive research on LAB fermentation, the regulation of metabolism during the growth in complex food-industry-relevant environments remains unclear. This study investigates the metabolic processes in Leuconostoc pseudomesenteroides DSM20193 during 24 h fermentation of BSG with and without 4% sucrose (w/w) supplementation, allowing in situ dextran synthesis. Besides dextran synthesis, the presence of sucrose led to faster acidification, especially due to the increased formation of acetic acid. Furthermore, differences in the utilisation of sucrose, fructose, glucose, and maltose and the formation of diverse oligosaccharides were observed. Transcriptome analysis comparing expression profiles during 0 h and 16 h growth in BSG with sucrose revealed differences in the expression of genes involved in carbohydrate utilisation pathways, including higher activity of sucrose and maltose metabolism and lower activity of metabolism related to alternative carbon sources. Transcription analysis of selected relevant genes in a time-course comparison between BSG with and without sucrose provided more detailed indications of responses of the metabolic network in this complex environment. This analysis provided a deeper understanding of the dynamic regulatory mechanism that drives sugar metabolism and dextran synthesis and how the presence of sucrose can alter the metabolic flux towards different fermentation products.

The species Saccharomyces uvarum and Saccharomyces kudriavzevii have gained popularity in recent decades due to their interesting oenological properties. However, although it plays a crucial role in yeast fermentation performance and compound synthesis, our understanding of nitrogen metabolism in these species remains limited. Therefore, we compared how three strains of Saccharomyces cerevisiae, Saccharomyces uvarum and Saccharomyces kudriavzevii use relevant nitrogen sources by combining quantitative analysis approaches based on isotopic tracing and modelling. The model we have developed aims to facilitate the calculation and interpretation of stable isotope data for other experiments, by providing easy visualisation of the results and predicting the kinetics of isotope incorporation beyond the sampling points. The three species exhibit significant variations in their nitrogen assimilation profile. They differ in the timing of uptake of ammonium, arginine and glutamine: Saccharomyces cerevisiae prefers glutamine, Saccharomyces kudriavzevii ammonium and Saccharomyces uvarum arginine. This contributes to a different pattern of nitrogen redistribution towards proteinogenic amino acids between strains at the start of the exponential phase, which fades on entering the stationary phase. Additionally, we found that the contribution of leucine and valine to isoamyl alcohol production varies between species; also, Saccharomyces kudriavzevii activates the synthesis of volatile compounds earlier.

We developed a sensitive and specific method based on recombinase-aided amplification (RAA) and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 13a (Cas13a). This method, named CRISPR-based Rapid and Efficient Test (CRISPRET), is designed for the early diagnosis of Influenza B (FluB) with the aim of shortening its transmission chain. We identified conserved regions in the Influenza B Virus (IBV) NS gene and designed forward and reverse primers along with crRNAs. We then established and optimised the reaction system, and Nucleic Acid Positive Reference Materials of IBV were used to evaluate the detection limit (DL) of CRISPRET. Additionally, we collected 257 clinical samples, comprising 127 samples from patients with IBV infection and 130 samples from healthy individuals, and subjected them to dual detection using CRISPRET and qPCR to evaluate the positive predictive value (PPV), negative predictive value (NPV), sensitivity and specificity of CRISPRET. We designed one forward primer, two reverse primers, and two crRNAs to establish and optimise the CRISPR ET. The method demonstrated the DL of 500 copies·μL⁻¹ when assisted by appropriate equipment. Despite requiring auxiliary equipment and a 30-min reaction, the CRISPR ET method enables the detection of IBV nucleic acid within approximately the first 5 min, achieving high sensitivity (100%), specificity (97.69%), PPV (97.69%) and NPV (100%), with a concordance rate of 98.83% to qPCR. CRISPRET offers a simple, field-applicable, one-step method for the rapid detection of IBV. It has strong potential for field-testing applications and intelligent integration into existing diagnostic systems.

Microbes govern our planet! The International Microbial Literacy Initiative (IMiLI) promotes global microbial literacy with free, open-access resources in multiple languages. Understanding microbes is key to sustainability and informed decision-making.

An upsurge of monkeypox disease (mpox) cases with clade I virus in Central Africa led WHO to declare a Public Health Emergency of International Concern for a second time shortly after the worldwide clade II mpox epidemic in 2022/3 among homosexual men. In the Democratic Republic of Congo (DRC), the annual incidence of clade I mpox, transmitted mostly from animal sources to children, increased 20-fold between 1980 and 2007; 60,000 mpox cases occurred between 2010 and 2023. The incidence again doubled between 2023 and 2024, showing a case fatality rate of 3.3%. A new clade Ib virus was detected in 2024 in eastern DRC where mostly adults were infected by heterosexual contact. Ib was recently introduced and showed a mutation spectrum of human-to-human transmission. Asymptomatic mpox infections, the release of infectious virus before symptom onset in a subgroup of cases, and superspreaders complicate containment measures during the 2022 epidemic. Isolation of cases until two consecutive negative PCR tests was recommended but necessitates cheap and rapid diagnostic tests which are in development. Sexual behavioural changes during the 2022 epidemic have contributed more to the curbing of the epidemic than vaccination. The smallpox vaccine Dryvax protected children exposed to clade I mpox in DRC in the 1980s. The attenuated third-generation smallpox Modified Vaccinia Ankara (MVA) vaccines and derivatives showed robust protection against clade IIb mpox during the 2022/3 epidemic in various study formats. Vaccine efficacy exceeding 75% was reported after two doses. mRNA in lipid-nanoparticle encoding surface proteins from extracellular enveloped and intracellular mature virions of monkeypox virus (MPXV) induced humoral and cellular immune responses that protected macaques against mpox disease with clade I and II viruses better than MVA. Only mixtures of monoclonal antibodies protected mice from mpox. The antiviral tecovirimat showed no efficacy in two clinical trials against clade I and II mpox.

The EU Green Deal prioritises the transformation of the chemical industry to a more environmentally sustainable model. This involves using microorganisms, such as Saccharomyces cerevisiae, to produce molecules more sustainably through biotechnological approaches. In this study, we demonstrate an example of serotonin production using S. cerevisiae as a cell factory, along with its optimisation and upscaling. To achieve this, we introduced two heterologous genes, the combination of tryptophan decarboxylase from Clostridium sporogenes (CsTDC) and tryptamine 5-hydroxylase from Oryza sativa (OsT5H), to complete the serotonin biosynthetic pathway using L-tryptophan (L-TRP) as a precursor. By modifying ARO4 to a feedback-resistant version (ARO4*), the flux of the shikimate pathway was significantly increased and serotonin production was achieved at levels up to 120 mg/L directly from the glucose source. After a medium optimisation, a final concentration of 80 g/L glucose and 300 mg/L of nitrogen resulted in better conditions for increasing serotonin titres. Using this medium in a 1 L bioreactor fermentation resulted in approximately 250 mg/L of serotonin. A targeted metabolomic study of the bioreactor growth medium identified potential bottlenecks in the serotonin-overproducing strain and future targets for increasing its titre. We have constructed a strain of S. cerevisiae that represents the first steps towards feasible industrial production of serotonin using a sustainable and environmentally friendly approach, paving the way for the development of similar biotechnological strategies in the future.

Microbes are essential in every environment, with an important role in the fight against climate change. Promoting microbiology literacy, especially among children, is crucial. Our Ibero-American consortium uses gamification to make science and microbiology education engaging, tested through outreach campaigns in Costa Rica, and promoting future campaigns across Latin America.

In many microbial biotechnology processes, biomass itself is not the product of interest, but rather targeted chemicals or proteins. In these processes, growth should be limited to direct more substrate to product and increase process yields. Under growth-limiting conditions, such as nutrient limitation, microorganisms, including the yeast Saccharomyces cerevisiae, activate a general stress response (GSR). Different hypotheses have been formulated for this activation, including a preparatory role for future stresses or a role in cellular protein density. Here we tested a third hypothesis: the GSR reduces the energy needed to maintain cellular homeostasis, also known as the maintenance energy requirement (MER). The impact of GSR on MER was investigated by assessing the effect of the absence of its key regulators, Msn2 and Msn4, on energy-substrate distribution and stress resistance. Chemostat and fed-batch cultures revealed significant increases in MER of up to 85% in the deletion strain compared to the parental strain. In contrast, maximal biomass yields, growth rates and morphology were unaffected. Our insights highlight an additional role of the GSR, namely saving cellular energy. As the MER is a key determinant of product yields and in process design, especially in low growth processes, our findings can help to optimise microbial bioprocesses.

Psilocybin, a tryptamine-derived alkaloid, has been granted Breakthrough Therapy designation by the U.S. FDA for treatment-resistant depression, underscoring its clinical importance. Therefore, sustainable and economic production is urgently needed. Manufacturing of psilocybin in Escherichia coli has drawn great attention. However, due to the low expression and activity of the eukaryotic cytochrome P450 enzyme PsiH in the psilocybin biosynthetic pathway, de novo synthesis of psilocybin in prokaryotic cells has been hampered. To overcome this dilemma, we herein demonstrated de novo synthesis of psilocybin in E. coli by constructing PsiH variants with N-terminal domain modifications and expressing the entire biosynthetic pathway at a concordantly low temperature. Improving the supply of precursor and engineering the P450 electron transfer chain resulted in a 33-fold increase in the titre of norbaeocystin (105.3 mg/L), a key intermediate of psilocybin biosynthesis, and a 17-fold increase in the titre of psilocybin (14 mg/L). Further enhancement of psilocybin production was achieved by converting norbaeocystin to psilocybin by overexpressing an extra copy of the methyltransferase gene psiM. Finally, 79.4 mg/L of psilocybin was produced by optimising flask fermentation conditions, a 100-fold improvement over the starting strain. Our work demonstrates the successful fungal P450 engineering to improve the catalytic activity in E. coli and will advance the sustainable production of the important antidepressant psilocybin in prokaryotic microbial cells.