Genome Mining of Fungal Unique Trichodiene Synthase-like Sesquiterpene Synthases.

Sesquiterpenoids served as an important source for natural product drug discovery. Although genome mining approaches have revealed numerous novel sesquiterpenoids and biosynthetic enzymes, the comprehensive landscape of fungal sesquiterpene synthases (STSs) remains elusive. In this study, 123 previously reported fungal STSs were subjected to phylogenetic analysis, resulting in the identification of a fungi-specific STS family known as trichodiene synthase-like sesquiterpene synthases (TDTSs). Subsequently, the application of hidden Markov models allowed the discovery of 517 TDTSs from our in-house fungi genome library of over 400 sequenced genomes, and these TDTSs were defined into 79 families based on a sequence similarity network. Based on the novelty of protein sequences and the completeness of their biosynthetic gene clusters, 23 TDTS genes were selected for heterologous expression in Aspergillus oryzae. In total, 10 TDTSs were active and collectively produced 12 mono- and sesquiterpenes, resulting in the identification of the first chamipinene synthase, as well as the first fungi-derived cedrene, sabinene, and camphene synthases. Additionally, with the guidance of functionally characterized TDTSs, we found that TDTSs in Family 1 could produce bridged-cyclic sesquiterpenes, while those in Family 2 could synthesize spiro- and bridged-cyclic sesquiterpenes. Our research presents a new avenue for the genome mining of fungal sesquiterpenoids.

Comparative transcriptome analysis reveals the redirection of metabolic flux from cell growth to astaxanthin biosynthesis in Yarrowia lipolytica.

Engineering Yarrowia lipolytica to produce astaxanthin provides a promising route. Here, Y. lipolytica M2 producing a titer of 181 mg/L astaxanthin was isolated by iterative atmospheric and room-temperature plasma mutagenesis and diphenylamine-mediated screening. Interestingly, a negative correlation was observed between cell biomass and astaxanthin production. To reveal the underlying mechanism, RNA-seq analysis of transcriptional changes was performed in high producer M2 and reference strain M1, and a total of 1379 differentially expressed genes were obtained. Data analysis revealed that carbon flux was elevated through lipid metabolism, acetyl-CoA and mevalonate supply, but restrained through central carbon metabolism in strain M2. Moreover, upregulation of other pathways such as ATP-binding cassette transporter and thiamine pyrophosphate possibly provided more cofactors for carotenoid hydroxylase and relieved cell membrane stress caused by astaxanthin insertion. These results suggest that balancing cell growth and astaxanthin production may be important to promote efficient biosynthesis of astaxanthin in Y. lipolytica.

Expanding catalytic promiscuity of a bifunctional terpene synthase through a single mutation-induced change in hydrogen-bond network within the catalytic pocket.

Fungal bifunctional terpene synthases (BFTSs) catalyze the formation of numerous di-/sester-/tri-terpenes skeletons. However, the mechanism in controlling the cyclization pattern of terpene scaffolds is rarely deciphered for further application of tuning the catalytic promiscuity of terpene synthases for expanding the chemical space. In this study, we expanded the catalytic promiscuity of Fusarium oxysporum fusoxypene synthase (FoFS) by a single mutation at L89, leading to the production of three new sesterterpenes. Further computational analysis revealed that the reconstitution of the hydrogen-bond (H-bond) network of second-shell residues around the active site of FoFS influences the orientation of the aromatic residue W69 within the first-shell catalytic pocket. Thus, the dynamic orientation of W69 alters the carbocation transport, leading to the production of diverse ring system skeletons. These findings enhance our knowledge on understanding the molecular mechanisms, which could be applied on protein engineering terpene synthases on regulating the terpene skeletons.

Efficient expansion and delayed senescence of hUC-MSCs by microcarrier-bioreactor system.

BACKGROUND: Human umbilical cord mesenchymal stem cells (hUC-MSCs) are widely used in cell therapy due to their robust immunomodulatory and tissue regenerative capabilities. Currently, the predominant method for obtaining hUC-MSCs for clinical use is through planar culture expansion, which presents several limitations. Specifically, continuous cell passaging can lead to cellular aging, susceptibility to contamination, and an absence of process monitoring and control, among other limitations. To overcome these challenges, the technology of microcarrier-bioreactor culture was developed with the aim of ensuring the therapeutic efficacy of cells while enabling large-scale expansion to meet clinical requirements. However, there is still a knowledge gap regarding the comparison of biological differences in cells obtained through different culture methods. METHODS: We developed a culture process for hUC-MSCs using self-made microcarrier and stirred bioreactor. This study systematically compares the biological properties of hUC-MSCs amplified through planar culture and microcarrier-bioreactor systems. Additionally, RNA-seq was employed to compare the differences in gene expression profiles between the two cultures, facilitating the identification of pathways and genes associated with cell aging. RESULTS: The findings revealed that hUC-MSCs expanded on microcarriers exhibited a lower degree of cellular aging compared to those expanded through planar culture. Additionally, these microcarrier-expanded hUC-MSCs showed an enhanced proliferation capacity and a reduced number of cells in the cell cycle retardation period. Moreover, bioreactor-cultured cells differ significantly from planar cultures in the expression of genes associated with the cytoskeleton and extracellular matrix. CONCLUSIONS: The results of this study demonstrate that our microcarrier-bioreactor culture method enhances the proliferation efficiency of hUC-MSCs. Moreover, this culture method exhibits the potential to delay the process of cell aging while preserving the essential stem cell properties of hUC-MSCs.

Antioxidants Improve the Proliferation and Efficacy of hUC-MSCs against H2O2-Induced Senescence.

Human umbilical cord mesenchymal stem cells (hUC-MSCs) are broadly applied in clinical treatment due to convenient accessibility, low immunogenicity, and the absence of any ethical issues involved. However, the microenvironment of inflammatory tissues may cause oxidative stress and induce senescence in transplanted hUC-MSCs, which will further reduce the proliferation, migration ability, and the final therapeutic effects of hUC-MSCs. Beta-nicotinamide mononucleotide (NMN) and coenzyme Q10 (CoQ10) are famous antioxidants and longevity medicines that could reduce intracellular reactive oxygen species levels by different mechanisms. In this study, hUC-MSCs were treated in vitro with NMN and CoQ10 to determine if they could reduce oxidative stress caused by hydrogen peroxide (H2O2) and recover cell functions. The effects of NMN and CoQ10 on the cell proliferation, the mRNA levels of the inflammatory cytokine TNFα and the anti-inflammatory cytokine IL10, and the differentiation and cell migration ability of hUC-MSCs before and after H2O2 treatment were investigated. The findings revealed that NMN and CoQ10 reduced H2O2-induced senescence and increased hUC-MSCs' proliferation in the late phase as passage 12 and later. The TNFα mRNA level of hUC-MSCs induced by H2O2 was significantly decreased after antioxidant treatment. NMN and CoQ10 all reduced the adipogenic differentiation ability of hUC-MSCs. CoQ10 improved the chondrogenic differentiation ability of hUC-MSCs. Furthermore, NMN was found to significantly enhance the migration ability of hUC-MSCs. Transcriptomic analysis revealed that NMN and CoQ10 both increased DNA repair ability and cyclin expression and downregulated TNF and IL-17 inflammatory signaling pathways, thereby contributing to the proliferative promotion of senecent stem cells and resistance to oxidative stress. These findings suggest that antioxidants can improve the survival and efficacy of hUC-MSCs in stem cell therapy for inflammation-related diseases.

Construction of CAR-T cells targeting TM4SF1 and its anti-tumor capacity in ovarian cancer.

Ovarian cancer (OC) is the most lethal gynecological malignancy with a 5-year survival rate of 49.1% on average. In clinical practice, cytoreduction and chemotherapy remain the conventional treatment for advanced OC. However, the overall prognosis remains poor, which urges oncologists to develop new treatments. Chimeric antigen receptor (CAR)-T therapy as a branch of immunotherapy had gained a success in treating hematological malignancies. TM4SF1, a potential biomarker in many tumors, was validated highly expressed in ovarian cancer. Here we constructed a 3rd generation CAR-T agent targeting TM4SF1 to treat ovarian cancer. CAR-T cells showed a specific cytotoxicity against TM4SF1 positive tumor cell lines in vitro and repressed SKOV3-derived tumor growth in vivo. This is the first time reporting a CAR-T therapy targeting TM4SF1 in ovarian cancer. Our results suggested that TM4SF1 could be a very promising target in curing OC and showed the possibility of TM4SF1-based immunotherapy.

Enhancement of fatty acid degradation pathway promoted glucoamylase synthesis in Aspergillus niger.

BACKGROUND: Our recent multi-omics analyses of glucoamylase biosynthesis in Aspergillus niger (A. niger) suggested that lipid catabolism was significantly up-regulated during high-yield period under oxygen limitation. Since the catabolism of fatty acids can provide energy compounds such as ATP and important precursors such as acetyl-CoA, we speculated that enhancement of this pathway might be beneficial to glucoamylase overproduction. RESULTS: Based on previous transcriptome data, we selected and individually overexpressed five candidate genes involved in fatty acid degradation under the control of the Tet-on gene switch in A. niger. Overexpression of the fadE, fadA and cyp genes increased the final specific enzyme activity and total secreted protein on shake flask by 21.3 ~ 31.3% and 16.0 ~ 24.2%, respectively. And a better inducible effect by doxycycline was obtained from early logarithmic growth phase (18 h) than stationary phase (42 h). Similar with flask-level results, the glucoamylase content and total extracellular protein in engineered strains OE-fadE (overexpressing fadE) and OE-fadA (overexpressing fadA) on maltose-limited chemostat cultivation were improved by 31.2 ~ 34.1% and 35.1 ~ 38.8% compared to parental strain B36. Meanwhile, intracellular free fatty acids were correspondingly decreased by 41.6 ~ 44.6%. The metabolomic analysis demonstrated intracellular amino acids pools increased 24.86% and 18.49% in two engineered strains OE-fadE and OE-fadA compared to B36. Flux simulation revealed that increased ATP, acetyl-CoA and NADH was supplied into TCA cycle to improve amino acids synthesis for glucoamylase overproduction. CONCLUSION: This study suggested for the first time that glucoamylase production was significantly improved in A. niger by overexpression of genes fadE and fadA involved in fatty acids degradation pathway. Harnessing the intracellular fatty acids could be a strategy to improve enzyme production in Aspergillus niger cell factory.

Integrated pathway engineering and transcriptome analysis for improved astaxanthin biosynthesis in Yarrowia lipolytica.

Astaxanthin is a high value carotenoid with a broad range of commercial applications due to its superior antioxidant properties. In this study, ß-carotene-producing Yarrowia lipolytica XK17 constructed in the lab was employed for astaxanthin biosynthesis. The catalytic effects of ß-carotene ketolase CrtW and ß-carotene hydroxylase CrtZ from various species were investigated. The PspCrtW from Paracoccus sp. and HpCrtZ# from Haematococcus pluvialis were confirmed to be the best combination in converting ß-carotene. Several key bottlenecks in biomass and astaxanthin biosynthesis were effectively eliminated by optimizing the expression of the above enzymes and restoring uracil/leucine biosynthesis. In addition, the effects of astaxanthin biosynthesis on cell metabolism were investigated by integrated analysis of pathway modification and transcriptome information. After further optimization, strain DN30 was able to synthesize up to 730.3 mg/L astaxanthin in laboratory 5-L fermenter. This study provides a good metabolic strategy and a sustainable development platform for high-value carotenoid production.

Optimization of microbial cell factories for astaxanthin production: Biosynthesis and regulations, engineering strategies and fermentation optimization strategies.

The global market demand for natural astaxanthin is rapidly increasing owing to its safety, the potential health benefits, and the diverse applications in food and pharmaceutical industries. The major native producers of natural astaxanthin on industrial scale are the alga Haematococcus pluvialis and the yeast Xanthopyllomyces dendrorhous. However, the natural production via these native producers is facing challenges of limited yield and high cost of cultivation and extraction. Alternatively, astaxanthin production via metabolically engineered non-native microbial cell factories such as Escherichia coli, Saccharomyces cerevisiae and Yarrowia lipolytica is another promising strategy to overcome these limitations. In this review we summarize the recent scientific and biotechnological progresses on astaxanthin biosynthetic pathways, transcriptional regulations, the interrelation with lipid metabolism, engineering strategies as well as fermentation process control in major native and non-native astaxanthin producers. These progresses illuminate the prospects of producing astaxanthin by microbial cell factories on industrial scale.

Exploration and characterization of hypoxia-inducible endogenous promoters in Aspergillus niger.

Aspergillus niger is widely used for the efficient production of organic acids and enzyme preparations. However, this organism lacks basic genetic elements for dynamic control, especially inducible promoters that can respond to specific environmental signals. Since these are desirable for better adaptation of fermentation to large-scale industrial production, herein, we have identified the two first hypoxia-inducible promoters in A. niger, PsrbB and PfhbA. Their performance under high or low oxygen conditions was monitored using two reporter proteins, green fluorescent protein (EGFP) and ß-glucuronidase (GUS). For comparison, basal expression of the general strong promoter PgpdA was lower than PsrbB but higher than PfhbA. However, under hypoxia, both promoters showed higher expression than under hyperoxia, and these values were also higher than those observed for PgpdA. For PsrbB, strength under hypoxia was ~2-3 times higher than under hyperoxia (for PfhbA, 3-9 times higher) and ~2.5-5 times higher than for PgpdA (for PfhbA, 2-3 times higher). Promoter truncation analysis showed that the PsrbB fragment -1024 to -588 bp is the core region that determines hypoxia response. KEY POINTS: The first identification of two hypoxia-inducible promoters in A. niger is a promising tool for modulation of target genes under hypoxia. Two reporter genes revealed a different activity and responsiveness to hypoxia of PfhbA and PsrbB promoters, which is relevant for the development of dynamic metabolic regulation of A. niger fermentation. PsrbB promoter truncation and bioinformatics analysis is the foundation for further research.

Polyketide pesticides from actinomycetes.

Natural product derived pesticides have increased in popularity worldwide because of their high efficacy, eco-friendly nature and favorable safety profile. The development of polyketide pesticides from actinomycetes reflects this increase in popularity in the past decades. These pesticides, which include avermectins, spinosyns, polynactins, tetramycin and their analogues, have been successfully applied in crop protection. Moreover, the advance of biotechnology has led to continuous improvement in the discovery and production processes. In this review, we summarize these polyketide pesticides, their activities and provide insight into their development. We also discuss engineering strategies and the current status of industrial production for these pesticides. Given that actinomycetes are known to produce a wide range of bioactive secondary metabolites, the description of pesticide development and high yield strain improvement presented herein will facilitate further development of these valuable polyketide pesticides from actinomycetes.

Recent advances in biotechnology for marine enzymes and molecules.

The marine environment is the most biologically and chemically diverse habitat on Earth, and provides numerous marine-derived products, including enzymes and molecules, for industrial and pharmaceutical applications. Marine biotechnology provides important biological resources from marine habitat conservation to applied science. In recent years, advances in techniques in interdisciplinary research fields, including metabolic engineering and synthetic biology have significantly improved the production of marine-derived commodities. In this review, we outline the recent progress in the use or marine enzymes and molecules in biotechnology, including newly discovered products, function optimization of enzymes, and production improvement of small molecules.

Berberine reverses multidrug resistance in Candida albicans by hijacking the drug efflux pump Mdr1p.

Clinical use of antimicrobials faces great challenges from the emergence of multidrug-resistant pathogens. The overexpression of drug efflux pumps is one of the major contributors to multidrug resistance (MDR). Reversing the function of drug efflux pumps is a promising approach to overcome MDR. In the life-threatening fungal pathogen Candida albicans, the major facilitator superfamily (MFS) transporter Mdr1p can excrete many structurally unrelated antifungals, leading to MDR. Here we report a counterintuitive case of reversing MDR in C. albicans by using a natural product berberine to hijack the overexpressed Mdr1p for its own importation. Moreover, we illustrate that the imported berberine accumulates in mitochondria and compromises the mitochondrial function by impairing mitochondrial membrane potential and mitochondrial Complex I. This results in the selective elimination of Mdr1p overexpressed C. albicans cells. Furthermore, we show that berberine treatment can prolong the mean survival time of mice with blood-borne dissemination of Mdr1p overexpressed multidrug-resistant candidiasis. This study provides a potential direction of novel anti-MDR drug discovery by screening for multidrug efflux pump converters.

In vitro CRISPR/Cas9 system for genome editing of Aspergillus niger based on removable bidirectional selection marker AmdS.

We established an in vitro clustered regularly interspaced short palindromic repeats (CRISPR)-associated RNA-guided DNA endonucleases (Cas9) system to efficiently produce specific genome editing in Aspergillus niger, using a novel recyclable, bidirectional selection marker gene amdS without the need of prior production of an amdS mutant. The donor DNA plasmid consisted of amdS open reading frame, promoter, terminator, and directional repeats (DRs) flanking sequences. It was cotransformed with recombinant nuclease Cas9 and the sgRNA, which targets to the pigment gene olvA of A. niger strain CBS513.88. The positive olive transformants, other than the wild-type strain, were able to grow on the media containing acetamide as the sole nitrogen source and cesium chloride. Furthermore, culturing the transformants on media with fluoroacetamide and urea allowed a loop-out of the amdS expression cassette by recombining the flanking DRs. This study confirmed the facts that the endogenous amdS can be used as a dominant marker and that it can be removed by counter-selection in gene editing of A. niger. The proposed in vitro CRISPR/Cas9 method offers a powerful tool for marker-free genetic manipulation of filamentous fungi industrial-specific strains.

Engineering cofactor metabolism for improved protein and glucoamylase production in Aspergillus niger.

BACKGROUND: Nicotinamide adenine dinucleotide phosphate (NADPH) is an important cofactor ensuring intracellular redox balance, anabolism and cell growth in all living systems. Our recent multi-omics analyses of glucoamylase (GlaA) biosynthesis in the filamentous fungal cell factory Aspergillus niger indicated that low availability of NADPH might be a limiting factor for GlaA overproduction. RESULTS: We thus employed the Design-Build-Test-Learn cycle for metabolic engineering to identify and prioritize effective cofactor engineering strategies for GlaA overproduction. Based on available metabolomics and 13C metabolic flux analysis data, we individually overexpressed seven predicted genes encoding NADPH generation enzymes under the control of the Tet-on gene switch in two A. niger recipient strains, one carrying a single and one carrying seven glaA gene copies, respectively, to test their individual effects on GlaA and total protein overproduction. Both strains were selected to understand if a strong pull towards glaA biosynthesis (seven gene copies) mandates a higher NADPH supply compared to the native condition (one gene copy). Detailed analysis of all 14 strains cultivated in shake flask cultures uncovered that overexpression of the gsdA gene (glucose 6-phosphate dehydrogenase), gndA gene (6-phosphogluconate dehydrogenase) and maeA gene (NADP-dependent malic enzyme) supported GlaA production on a subtle (10%) but significant level in the background strain carrying seven glaA gene copies. We thus performed maltose-limited chemostat cultures combining metabolome analysis for these three isolates to characterize metabolic-level fluctuations caused by cofactor engineering. In these cultures, overexpression of either the gndA or maeA gene increased the intracellular NADPH pool by 45% and 66%, and the yield of GlaA by 65% and 30%, respectively. In contrast, overexpression of the gsdA gene had a negative effect on both total protein and glucoamylase production. CONCLUSIONS: This data suggests for the first time that increased NADPH availability can indeed underpin protein and especially GlaA production in strains where a strong pull towards GlaA biosynthesis exists. This data also indicates that the highest impact on GlaA production can be engineered on a genetic level by increasing the flux through the pentose phosphate pathway (gndA gene) followed by engineering the flux through the reverse TCA cycle (maeA gene). We thus propose that NADPH cofactor engineering is indeed a valid strategy for metabolic engineering of A. niger to improve GlaA production, a strategy which is certainly also applicable to the rational design of other microbial cell factories.

Improving cytosolic aspartate biosynthesis increases glucoamylase production in Aspergillus niger under oxygen limitation.

BACKGROUND: Glucoamylase is one of the most industrially applied enzymes, produced by Aspergillus species, like Aspergillus niger. Compared to the traditional ways of process optimization, the metabolic engineering strategies to improve glucoamylase production are relatively scarce. RESULTS: In the previous study combined multi-omics integrative analysis and amino acid supplementation experiment, we predicted four amino acids (alanine, glutamate, glycine and aspartate) as the limited precursors for glucoamylase production in A. niger. To further verify this, five mutants namely OE-ala, OE-glu, OE-gly, OE-asp1 and OE-asp2, derived from the parental strain A. niger CBS 513.88, were constructed respectively for the overexpression of five genes responsible for the biosynthesis of the four kinds of amino acids (An11g02620, An04g00990, An05g00410, An04g06380 and An16g05570). Real-time quantitative PCR revealed that all these genes were successfully overexpressed at the mRNA level while the five mutants exhibited different performance in glucoamylase production in shake flask cultivation. Notably, the results demonstrated that mutant OE-asp2 which was constructed for reinforcing cytosolic aspartate synthetic pathway, exhibited significantly increased glucoamylase activity by 23.5% and 60.3% compared to CBS 513.88 in the cultivation of shake flask and the 5 L fermentor, respectively. Compared to A. niger CBS 513.88, mutant OE-asp2 has a higher intracellular amino acid pool, in particular, alanine, leucine, glycine and glutamine, while the pool of glutamate was decreased. CONCLUSION: Our study combines the target prediction from multi-omics analysis with the experimental validation and proves the possibility of increasing glucoamylase production by enhancing limited amino acid biosynthesis. In short, this systematically conducted study will surely deepen the understanding of resources allocation in cell factory and provide new strategies for the rational design of enzyme production strains.

Comparative genomics of the aconidial Aspergillus niger strain LDM3 predicts genes associated with its high protein secretion capacity.

Aspergillus niger is widely used as a cell factory for homologous and heterologous protein production. As previous studies reported that reduced sporulation favors protein secretion in A. niger, in this study, we conducted a comparative genomic analysis of the non-sporulating industrially exploited A. niger strain LDM3 in China and the reference protein secretion strain CBS 513.88 to predict the key genes that might define the genetic basis of LDM3's high protein-producing potential in silico. After sequencing using a hybrid approach combining Illumina and PacBio sequencing platforms, a high-quality genome sequence of LDM3 was obtained which harbors 11,209 open reading frames (ORFs). LDM3 exhibits large chromosomal rearrangements in comparison to CBS 513.88. An alignment of the two genome sequences revealed that the majority of the 457 ORFs uniquely present in LDM3 possessed predicted functions in redox pathways, protein transport, and protein modification processes. In addition, bioinformatic analyses revealed the presence of 656 ORFs in LDM3 with non-synonymous mutations encoding for proteins related to protein translation, protein modification, protein secretion, metabolism, and energy production. We studied the impact of two of these on protein production in the established lab strain N402. Both tupA and prpA genes were selected because available literature suggested their involvement in asexual sporulation of A. niger. Our co-expression network analysis supportively predicted the role of tupA in protein secretion and the role of prpA in energy generation, respectively. By knockout experiments, we showed that the ΔtupA mutant displayed reduced sporulation (35%) accompanied by higher total protein secretion (65%) compared to its parental strain. Such an effect was, however, not observed in the ΔprpA mutant.

Genomics course design and combined teaching strategy to enhance learning initiatives in classroom.

Genomics is a core course for bioscience-majored undergraduates in many universities. However, the teaching of genomics faces many challenges such as the need for rapid updating of related knowledge, content overlap between genomics and molecular biology, lack of motivation of students in the traditional passive classroom-learning model, the difficulty of presenting omics data analysis processes through lectures, and the challenges of developing the research abilities of senior students. Here we report our updated genomics course design and the teaching practices we used to address these issues, including: use of the presentation-assimilation-discussion (PAD) pedagogy and opportunities for students to practice real data analysis and intensive reading of research papers. The resultant positive outcomes and problems are also discussed. © 2019 International Union of Biochemistry and Molecular Biology, 47(6):632-637, 2019.

Multi-omics integrative analysis with genome-scale metabolic model simulation reveals global cellular adaptation of Aspergillus niger under industrial enzyme production condition.

Oxygen limitation is regarded as a useful strategy to improve enzyme production by mycelial fungus like Aspergillus niger. However, the intracellular metabolic response of A. niger to oxygen limitation is still obscure. To address this, the metabolism of A. niger was studied using multi-omics integrated analysis based on the latest GEMs (genome-scale metabolic model), including metabolomics, fluxomics and transcriptomics. Upon sharp reduction of the oxygen supply, A. niger metabolism shifted to higher redox level status, as well as lower energy supply, down-regulation of genes for fatty acid synthesis and a rapid decrease of the specific growth rate. The gene expression of the glyoxylate bypass was activated, which was consistent with flux analysis using the A. niger GEMs iHL1210. The increasing flux of the glyoxylate bypass was assumed to reduce the NADH formation from TCA cycle and benefit maintenance of the cellular redox balance under hypoxic conditions. In addition, the relative fluxes of the EMP pathway were increased, which possibly relieved the energy demand for cell metabolism. The above multi-omics integrative analysis provided new insights on metabolic regulatory mechanisms of A. niger associated with enzyme production under oxygen-limited condition, which will benefit systematic design and optimization of the A. niger microbial cell factory.

Integrated analysis of the yeast NADPH-regulator Stb5 reveals distinct differences in NADPH requirements and regulation in different states of yeast metabolism.

The Saccharomyces cerevisiae transcription factor (TF) Stb5 is known to be involved in regulating NADPH generation. We explored its role by combining DNA binding studies with transcriptome analysis at four environmental conditions that were selected to cover a range of different metabolic states. Using ChIP-exo, DNA binding targets of Stb5 were found to confirm many previously proposed binding targets, in particular genes encoding enzymes involved in NADPH generation and the pentose-phosphate (PP) pathway. Transcriptome analysis of an STB5 deletion strain revealed transcriptional changes in direct regulation targets of Stb5, including several PP pathway genes as well as additional novel regulatory targets, but interestingly not including the proposed PP pathway flux controlling enzyme Zwf1. Consistently, NADPH levels were found to decrease significantly with STB5 deletion in cultures with aerobic, glucose metabolism. We also found reduced growth for the STB5 deletion strain in similar conditions as those with reduced NADPH levels, supporting a role for Stb5 in NADPH generation through the PP pathway. We finally explored the flux distribution by genome scale modelling simulations and found a decreased flux in both NADPH generating as well as consuming reactions in the STB5 deletion strain.