Genome-scale metabolic model (GSMM) is a powerful tool for the study of cellular metabolic characteristics. With the development of multi-omics measurement techniques in recent years, new methods ...that integrating multi-omics data into the GSMM show promising effects on the predicted results. It does not only improve the accuracy of phenotype prediction but also enhances the reliability of the model for simulating complex biochemical phenomena, which can promote theoretical breakthroughs for specific gene target identification or better understanding the cell metabolism on the system level. Based on the basic GSMM model iHL1210 of Aspergillus niger, we integrated large-scale enzyme kinetics and proteomics data to establish a GSMM based on enzyme constraints, termed a GEM with Enzymatic Constraints using Kinetic and Omics data (GECKO). The results show that enzyme constraints effectively improve the model's phenotype prediction ability, and extended the model's potential to guide target gene identification through predicting metabolic phenotype changes of A. niger by simulating gene knockout. In addition, enzyme constraints significantly reduced the solution space of the model, i.e., flux variability over 40.10% metabolic reactions were significantly reduced. The new model showed also versatility in other aspects, like estimating large-scale formula omitted values, predicting the differential expression of enzymes under different growth conditions. This study shows that incorporating enzymes' abundance information into GSMM is very effective for improving model performance with A. niger. Enzyme-constrained model can be used as a powerful tool for predicting the metabolic phenotype of A. niger by incorporating proteome data. In the foreseeable future, with the fast development of measurement techniques, and more precise and rich proteomics quantitative data being obtained for A. niger, the enzyme-constrained GSMM model will show greater application space on the system level.
•Salt stress could enhance the lipid synthesis in heterotrophic C. protothecoides.•Effect of salt stress was better than osmotic stress at the same osmolarity level.•ROS production caused by the ...stress condition may mediate lipid accumulation.•Mechanisms of lipid accumulation were revealed by the multi-level analysis.
Salt stress as an effective stress factor that could improve the lipid content and lipid yield of glucose in the heterotrophic culture cells of Chlorella protothecoides was demonstrated in this study. The highest lipid content of 41.2% and lipid yield of 185.8mg/g were obtained when C. protothecoides was stressed under 30g/L NaCl condition at its late logarithmic growth phase. Moreover, the effects of salt and osmotic stress on lipid accumulation were comparatively analyzed, and it was found that the effects of NaCl and KCl stress had no significant differences at the same osmolarity level of 1150mOsm/kg with lipid contents of 41.7 and 40.8% as well as lipid yields of 192.9 and 186.8mg/g, respectively, whereas these results were obviously higher than those obtained under the iso-osmotic glycerol and sorbitol stresses. Furthermore, basing on the multi-level analysis of oxidative response, key enzyme activity and biochemical alteration, the superior performance of salt stress driving lipid over-synthesis was probably ascribed to the more ROS production as a result of additional ion effect besides the osmotic effect, subsequently mediating the alteration from carbohydrate storage to lipid accumulation in signal transduction process of C. protothecoides.
Metabolome defines a set of metabolites present in a biological sample, which provides an immediate and dynamic recording of microbes in response to genetic and/or environmental perturbations. In ...recent years, metabolomics in combination with other omics diagnostic tools such as genomics, transcriptomics and proteomics is focused on addressing open biological questions that accelerate our understanding of the system as a whole and boost the use of systems metabolic engineering tools in industrial settings. In this review article, we summarize the applications of metabolomics to industrial microbial fermentations with respect to the bulk production of organic acids, amino acids, enzymes, antibiotics and therapeutic proteins. In addition, future prospects regarding metabolomics-assisted research are provided.
The grand challenge now and in the near future for the pharmaceutical industry is how to efficiently improve R&D productivity. Currently, the approval rate of the entire clinical drug development ...process is extremely low, and the high attrition in the phase I clinical trial is up to 95%; 67% and 33% of all drugs that enter Phase II and Phase III clinical trials fail to transit into the next stage, respectively. To achieve a higher success rate in clinical trials, developing efficient drug screening method based on more in vivo like tumor tissue is an urgent need to predict the toxicity and efficacy of candidate drugs. In comparison to 2D planar tumor model, the 3D multicellular tumor spheroid (MTS) can better simulate the spatial structure, hypoxia and nutrient gradient, extracellular matrix (ECM) deposition and drug resistance mechanism of tumor in vivo. Thus, such model can be applied for high-throughput drug screening and evaluation, and also can be utilized to initiate a series of fundamental research areas regarding oncogenesis, tumor progression and invasion, pharmacokinetics, drug metabolism, gene therapy and immune mechanism. This review article discusses the abnormal metabolism of cancer cells and highlights the potential role of MTSs as being used as efficient preclinical models. Also, the key features and preparation protocols of MTSs as well as the tools and techniques used for their analysis were summarized and the application of 3D tumor spheroid in specific drug screening and in the elucidation of drug resistance mechanism was also provided. Despite the great knowledge gap within biological sciences and bioengineering, the grand blueprint for adaptable stirred-tank culture strategies for large-scale production of MTSs is envisioned.
•First-time studied the effect of A. niger morphology on gluconic acid (GA) production.•Dispersed mycelia was proposed for GA production by online visualization methods.•Oxygen transfer between the ...media and mycelia was improved by using dispersed mycelia.•The average GA production rate had 1.7-fold increase in dispersed fermentation.•The yield of GA fermentation with dispersed seed is 3.1% higher than the best reported data.
Effects of seed morphology of Aspergillus niger (A. niger) AN151 on gluconic acid (GA) biosynthesis were investigated in 50-L fermenters. Online physiological parameters showed that the optimum seed morphology for GA biosynthesis was dispersed pattern rather than pellets which were widely used in industrial production. The highest overall yield of 1.051±0.012 (gg−1) was achieved using dispersed seed in shorter fermentation time (14.5h) with an initial glucose solution of 330gL−1, and was 3.1% higher than the best reported data. And the average GA production rate under dispersed fermentation (21.0±0.9gL−1h−1) was 73.6% and 39.1% higher than those with large and small pellets, respectively. The fermentation results showed that conversion of mycelial morphology from pellets to dispersed state had negligible impact on viscosity of fermentation broth at low biomass concentration (1.7gL−1), while coefficient of volumetric oxygen transfer (kLa) improved more than 13%, suggesting that the promotion of oxygen transfer rate (OTR) between liquid medium and mycelia greatly increased GA production. Therefore, controlling the morphology of A. niger is an effective method for improving industrial GA production.
In the recombinant protein market with broad economic value, the rapid development of synthetic biology has made it necessary to construct an efficient exocrine expression system for the different ...heterologous proteins. Yarrowia lipolytica possesses unique advantages in nascent protein transport and glycosylation modification, so it can serve as a potential protein expression platform. Although the Po1 series derived from W29 is often used for the expression of the various heterologous proteins, the ability of W29 to secrete proteins has not been verified and the Po1 series has been found to be not convenient for further gene editing. A total of 246 Y. lipolytica strains were evaluated for their secretory capacity through performing high-throughput screening in 48-well plate. Thereafter, following two rounds of shake flask re-screening, a high-secreting protein starting strain DBVPG 5851 was obtained. Subsequently, combined with the extracellular protein types and relative abundance information provided by the secretome of the starting strain, available chassis cell for heterologous protein expression were preliminarily constructed, and it was observed that the most potential signal peptide was derived from YALI0D20680g. This study offers a novel perspective on the diversification of Y. lipolytica host cells for the heterologous protein expression and provides significant basis for expanding the selection space of signal peptide tools in the future research.
Hexanol–butanol–ethanol fermentation from syngas by
Clostridium carboxidivorans
P7 is a promising route for biofuel production. However, bacterial agglomeration in the culture of 37 °C severely ...hampers the accumulation of biomass and products. To investigate the effect of culture temperature on biomass growth and higher-alcohol production,
C. carboxidivorans
P7 was cultivated at both constant and two-step temperatures in the range from 25 to 37 °C. Meanwhile, Tween-80 and saponin were screened out from eight surfactants to alleviate agglomeration at 37 °C. The results showed that enhanced higher-alcohol production was contributed mainly by the application of two-step temperature culture rather than the addition of anti-agglomeration surfactants. Furthermore, comparative transcriptome revealed that although 37 °C promoted high expression of genes involved in the Wood–Ljungdahl pathway, genes encoding enzymes catalyzing acyl-condensation reactions associated with higher-alcohol production were highly expressed at 25 °C. This study gained greater insight into temperature-effect mechanism on syngas fermentation by
C. carboxidivorans
P7.
Chlorogenic acids (CGAs) are bioactive compounds widely used in the food, pharmaceutical, and cosmetic industries.
is an important economic crop, and its suspension cells are rich in CGAs. However, ...little is known about the biosynthesis and regulation of CGAs in
cells. This study first elucidated the regulatory mechanism of CGA biosynthesis in methyl jasmonate (MeJA)-treated
cells and the role of the MeJA-responsive
(
) gene in enhancing their CGA accumulation. Firstly, temporal changes in intracellular metabolites showed that MeJA increased the intracellular CGA content up to 1.61-fold to 100.23 mg·g
. Meanwhile, 31 primary metabolites showed significant differences, with 6 precursors related to increasing CGA biosynthesis. Secondly, the transcriptome data revealed 3637 new genes previously unannotated in the
genome and 3653 differentially expressed genes. The genes involved in the plant signaling pathway and the biosynthesis of CGAs and their precursors showed a general up-regulation, especially the
gene family, which ultimately promoted CGA biosynthesis. Thirdly, the expression of a newly annotated and MeJA-responsive
gene (
, CtNewGene_3476) was demonstrated to be positively correlated with CGA accumulation in the cells, and transient overexpression of
enhanced CGA accumulation in tobacco. Finally, in vitro catalysis kinetics and molecular docking simulations revealed the ability and mechanism of the
HCT protein to bind to various substrates and catalyze the formation of four hydroxycinnamic esters, including CGAs. These findings strengthened our understanding of the regulatory mechanism of CGA biosynthesis, thereby providing theoretical support for the efficient production of CGAs.
The two-component system “AfsQ1/Q2” plays a crucial role to activate the production of antibiotics ACT, RED, and CDA through directly binding the promoters of pathway-specific activator genes
...actII-ORF4
,
redZ
, and
cdaR
respectively when grown under glutamate-supplemented minimal medium in
Streptomyces coelicolor
. In this report, we demonstrated that the RspA1/A2 (a homologous protein of two-component system AfsQ1/Q2) plays a regulatory role in salinomycin biosynthesis in
Streptomyces albus
. Gene deletion and complementation experiments showed that the RspA1/A2 promoted salinomycin production but inhibited cell growth when cultured in YMG medium supplemented with 3% soybean oil. More importantly, RspA1/A2 strengthens salinomycin biosynthesis by directly affecting the transcription of the pathway-specific activator gene
slnR
. Meanwhile, RspA1/A2 plays a negative role in the regulation of nitrogen assimilation and urea decarboxylation by interacting with the promoters of genes
gdhA
,
glnA
,
amtB
, and
SLNWT_1828/1829
. Gene
sigW
is located downstream of
rspA1/A2
and encodes an extracytoplasmic function sigma factor. Moreover, it negatively regulates the salinomycin biosynthesis and promotes cell growth, which antagonizes the function of RspA1/A2. In short, these useful findings are proved helpful to enrich the understanding of the regulatory pathways of antibiotic biosynthesis by an ECF σ factor-TCS signal transduction system in
Streptomyces
.
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