Microbial Cell Factories最新文献

Background: The gram-positive bacterium Bacillus subtilis is widely used for industrial enzyme production. Its ability to secrete a wide range of enzymes into the extracellular medium especially facilitates downstream processing since cell disruption is avoided. Although various heterologous enzymes have been successfully secreted with B. subtilis, the secretion of cytoplasmic enzymes with high molecular weight is challenging. Only a few studies report on the secretion of cytoplasmic enzymes with a molecular weight > 100 kDa.

Results: In this study, the cytoplasmic and 120 kDa β-galactosidase of Paenibacillus wynnii (β-gal-Pw) was expressed and secreted with B. subtilis SCK6. Different strategies were focused on to identify the best secretion conditions. Tailormade codon-optimization of the β-gal-Pw gene led to an increase in extracellular β-gal-Pw production. Consequently, the optimized gene was used to test four signal peptides and two promoters in different combinations. Differences in extracellular β-gal-Pw activity between the recombinant B. subtilis strains were observed with the successful secretion being highly dependent on the specific combination of promoter and signal peptide used. Interestingly, signal peptides of both the general secretory- and the twin-arginine translocation pathway mediated secretion. The highest extracellular activity of 55.2 ± 6 µkat/L_culture was reached when secretion was mediated by the PhoD signal peptide and expression was controlled by the P_AprE promoter. Production of extracellular β-gal-Pw was further enhanced 1.4-fold in a bioreactor cultivation to 77.5 ± 10 µkat/L_culture with secretion efficiencies of more than 80%.

Conclusion: For the first time, the β-gal-Pw was efficiently secreted with B. subtilis SCK6, demonstrating the potential of this strain for secretory production of cytoplasmic, high molecular weight enzymes.

There is increasing evidence that probiotic and commensal bacteria play a role in substrate metabolism, energy harvesting and intestinal homeostasis, and may exert immunomodulatory activities on human health. In addition, recent research suggests that these microorganisms interact with vitamins and minerals, promoting intestinal and metabolic well-being while producing vital microbial metabolites such as short-chain fatty acids (SCFAs). In this regard, there is a flourishing field exploring the intricate dynamics between vitamins, minerals, SCFAs, and commensal/probiotic interactions. In this review, we summarize some of the major hypotheses beyond the mechanisms by which commensals/probiotics impact gut health and their additional effects on the absorption and metabolism of vitamins, minerals, and SCFAs. Our analysis includes comprehensive review of existing evidence from preclinical and clinical studies, with particular focus on the potential interaction between commensals/probiotics and micronutrients. Finally, we highlight knowledge gaps and outline directions for future research in this evolving field.

Background: In vitro expression involves the utilization of the cellular transcription and translation machinery in an acellular context to produce one or more proteins of interest and has found widespread application in synthetic biology and in pharmaceutical biomanufacturing. Most in vitro expression systems available are active at moderate temperatures, but to screen large libraries of natural or artificial genetic diversity for highly thermostable enzymes or enzyme variants, it is instrumental to enable protein synthesis at high temperatures.

Objectives: Develop an in vitro expression system operating at high temperatures compatible with enzymatic assays and with technologies that enable ultrahigh-throughput protein expression in reduced volumes, such as microfluidic water-in-oil (w/o) droplets.

Results: We produced cell-free extracts from Thermus thermophilus for in vitro translation including thermostable enzymatic cascades for energy regeneration and a moderately thermostable RNA polymerase for transcription, which ultimately limited the temperature of protein synthesis. The yield was comparable or superior to other thermostable in vitro expression systems, while the preparation procedure is much simpler and can be suited to different Thermus thermophilus strains. Furthermore, these extracts have enabled in vitro expression in microfluidic droplets at high temperatures for the first time.

Conclusions: Cell-free extracts from Thermus thermophilus represent a simpler alternative to heavily optimized or pure component thermostable in vitro expression systems. Moreover, due to their compatibility with droplet microfluidics and enzyme assays at high temperatures, the reported system represents a convenient gateway for enzyme screening at higher temperatures with ultrahigh-throughput.

Background: Microbially induced calcium carbonate precipitation has been extensively researched for geoengineering applications as well as diverse uses within the built environment. Bacteria play a crucial role in producing calcium carbonate minerals, via enzymes including carbonic anhydrase-an enzyme with the capability to hydrolyse CO₂, commonly employed in carbon capture systems. This study describes previously uncharacterised carbonic anhydrase enzyme sequences capable of sequestering CO2 and subsequentially generating CaCO₃ biominerals and suggests a route to produce carbon negative cementitious materials for the construction industry.

Results: Here, Bacillus subtilis was engineered to recombinantly express previously uncharacterised carbonic anhydrase enzymes from Bacillus megaterium and used as a whole cell catalyst allowing this novel bacterium to sequester CO₂ and convert it to calcium carbonate. A significant decrease in CO₂ was observed from 3800 PPM to 820 PPM upon induction of carbonic anhydrase and minerals recovered from these experiments were identified as calcite and vaterite using X-ray diffraction. Further experiments mixed the use of this enzyme (as a cell free extract) with Sporosarcina pasteurii to increase mineral production whilst maintaining a comparable level of CO₂ sequestration.

Conclusion: Recombinantly produced carbonic anhydrase successfully sequestered CO₂ and converted it into calcium carbonate minerals using an engineered microbial system. Through this approach, a process to manufacture cementitious materials with carbon sequestration ability could be developed.

Background: White-rot fungi are known to naturally produce high quantities of laccase, which exhibit commendable stability and catalytic efficiency. However, their laccase production does not meet the demands for industrial-scale applications. To address this limitation, it is crucial to optimize the conditions for laccase production. However, the regulatory mechanisms underlying different conditions remain unclear. This knowledge gap hinders the cost-effective application of laccases.

Results: In this study, we utilized transcriptomic and metabolomic data to investigate a promising laccase producer, Cerrena unicolor 87613, cultivated with fructose as the carbon source. Our comprehensive analysis of differentially expressed genes (DEGs) and differentially abundant metabolites (DAMs) aimed to identify changes in cellular processes that could affect laccase production. As a result, we discovered a complex metabolic network primarily involving carbon metabolism and amino acid metabolism, which exhibited contrasting changes between transcription and metabolic patterns. Within this network, we identified five biomarkers, including succinate, serine, methionine, glutamate and reduced glutathione, that played crucial roles in co-determining laccase production levels.

Conclusions: Our study proposed a complex metabolic network and identified key biomarkers that determine the production level of laccase in the commercially promising Cerrena unicolor 87613. These findings not only shed light on the regulatory mechanisms of carbon sources in laccase production, but also provide a theoretical foundation for enhancing laccase production through strategic reprogramming of metabolic pathways, especially related to the citrate cycle and specific amino acid metabolism.

Background: Recombinant peptide production in Escherichia coli provides a sustainable alternative to environmentally harmful and size-limited chemical synthesis. However, in-vivo production of disulfide-bonded peptides at high yields remains challenging, due to degradation by host proteases/peptidases and the necessity of translocation into the periplasmic space for disulfide bond formation.

Results: In this study, we established an expression system for efficient and soluble production of disulfide-bonded peptides in the periplasm of E. coli. We chose model peptides with varying complexity (size, structure, number of disulfide bonds), namely parathyroid hormone 1-84, somatostatin 1-28, plectasin, and bovine pancreatic trypsin inhibitor (aprotinin). All peptides were expressed without and with the N-terminal, low molecular weight CASPON™ tag (4.1 kDa), with the expression cassette being integrated into the host genome. During BioLector™ cultivations at microliter scale, we found that most of our model peptides can only be sufficiently expressed in combination with the CASPON™ tag, otherwise expression was only weak or undetectable on SDS-PAGE. Undesired degradation by host proteases/peptidases was evident even with the CASPON™ tag. Therefore, we investigated whether degradation happened before or after translocation by expressing the peptides in combination with either a co- or post-translational signal sequence. Our results suggest that degradation predominantly happened after the translocation, as degradation fragments appeared to be identical independent of the signal sequence, and expression was not enhanced with the co-translational signal sequence. Lastly, we expressed all CASPON™-tagged peptides in two industry-relevant host strains during C-limited fed-batch cultivations in bioreactors. We found that the process performance was highly dependent on the peptide-host-combination. The titers that were reached varied between 0.6-2.6 g L^-1, and exceeded previously published data in E. coli. Moreover, all peptides were shown by mass spectrometry to be expressed to completion, including full formation of disulfide bonds.

Conclusion: In this work, we demonstrated the potential of the CASPON™ technology as a highly efficient platform for the production of soluble peptides in the periplasm of E. coli. The titers we show here are unprecedented whenever parathyroid hormone, somatostatin, plectasin or bovine pancreatic trypsin inhibitor were produced in E. coli, thus making our proposed upstream platform favorable over previously published approaches and chemical synthesis.

Background: Optically active D-amino acids are widely used as intermediates in the synthesis of antibiotics, insecticides, and peptide hormones. Currently, the two-enzyme cascade reaction is the most efficient way to produce D-amino acids using enzymes DHdt and DCase, but DCase is susceptible to heat inactivation. Here, to enhance the enzymatic activity and thermal stability of DCase, a rational design software "Feitian" was developed based on k_cat prediction using the deep learning approach.

Results: According to empirical design and prediction of "Feitian" software, six single-point mutants with high k_cat value were selected and successfully constructed by site-directed mutagenesis. Out of six, three mutants (Q4C, T212S, and A302C) showed higher enzymatic activity than the wild-type. Furthermore, the combined triple-point mutant DCase-M3 (Q4C/T212S/A302C) exhibited a 4.25-fold increase in activity (29.77 ± 4.52 U) and a 2.25-fold increase in thermal stability as compared to the wild-type, respectively. Through the whole-cell reaction, the high titer of D-HPG (2.57 ± 0.43 mM) was produced by the mutant Q4C/T212S/A302C, which was about 2.04-fold of the wild-type. Molecular dynamics simulation results showed that DCase-M3 significantly enhances the rigidity of the catalytic site and thus increases the activity of DCase-M3.

Conclusions: In this study, an efficient rational design software "Feitian" was successfully developed with a prediction accuracy of about 50% in enzymatic activity. A triple-point mutant DCase-M3 (Q4C/T212S/A302C) with enhanced enzymatic activity and thermostability was successfully obtained, which could be applied to the development of a fully enzymatic process for the industrial production of D-HPG.

Background: Type I interferons (IFN-I)-a group of cytokines with immunomodulatory, antiproliferative, and antiviral properties-are widely used as therapeutics for various cancers and viral diseases. Since IFNs are proteins, they are highly susceptible to degradation by proteases and by hydrolysis in the strong acid environment of the stomach, and they are therefore administered parenterally. In this study, we examined whether the intestinal bacterium, enteropathogenic Escherichia coli (EPEC), can be exploited for oral delivery of IFN-Is. EPEC survives the harsh conditions of the stomach and, upon reaching the small intestine, expresses a type III secretion system (T3SS) that is used to translocate effector proteins across the bacterial envelope into the eukaryotic host cells.

Results: In this study, we developed an attenuated EPEC strain that cannot colonize the host but can secrete functional human IFNα2 variant through the T3SS. We found that this bacteria-secreted IFN exhibited antiproliferative and antiviral activities similar to commercially available IFN.

Conclusion: These findings present a potential novel approach for the oral delivery of IFN via secreting bacteria.

Background: Syringic acid (SA) is a high-value natural compound with diverse biological activities and wide applications, commonly found in fruits, vegetables, and herbs. SA is primarily produced through chemical synthesis, nonetheless, these chemical methods have many drawbacks, such as considerable equipment requirements, harsh reaction conditions, expensive catalysts, and numerous by-products. Therefore, in this study, a novel biotransformation route for SA production was designed and developed by using engineered whole cells.

Results: An O-methyltransferase from Desulfuromonas acetoxidans (DesAOMT), which preferentially catalyzes a methyl transfer reaction on the meta-hydroxyl group of catechol analogues, was identified. The whole cells expressing DesAOMT can transform gallic acid (GA) into SA when S-adenosyl methionine (SAM) is used as a methyl donor. We constructed a multi-enzyme cascade reaction in Escherichia coli, containing an endogenous shikimate kinase (AroL) and a chorismate lyase (UbiC), along with a p-hydroxybenzoate hydroxylase mutant (PobA^**) from Pseudomonas fluorescens, and DesAOMT; SA was biosynthesized from shikimic acid (SHA) by using whole cells catalysis. The metabolic system of chassis cells also affected the efficiency of SA biosynthesis, blocking the chorismate metabolism pathway improved SA production. When the supply of the cofactor NADPH was optimized, the titer of SA reached 133 μM (26.2 mg/L).

Conclusion: Overall, we designed a multi-enzyme cascade in E. coli for SA biosynthesis by using resting or growing whole cells. This work identified an O-methyltransferase (DesAOMT), which can catalyze the methylation of GA to produce SA. The multi-enzyme cascade containing four enzymes expressed in an engineered E. coli for synthesizing of SA from SHA. The metabolic system of the strain and biotransformation conditions influenced catalytic efficiency. This study provides a new green route for SA biosynthesis.