Combinatorial metabolic engineering of Bacillus subtilis for de novo production of polymyxin B.

Polymyxin is a lipopeptide antibiotic that is effective against multidrug-resistant Gram-negative bacteria. However, its clinical development is limited due to low titer and the presence of homologs. To address this, the polymyxin gene cluster was integrated into Bacillus subtilis, and sfp from Paenibacillus polymyxa was expressed heterologously, enabling recombinant B. subtilis to synthesize polymyxin B. Regulating NRPS domain inhibited formation of polymyxin B2 and B3. The production of polymyxin B increased to 329.7 mg/L by replacing the native promoters of pmxA, pmxB, and pmxE with PfusA, C2up, and PfusA, respectively. Further enhancement in this production, up to 616.1 mg/L, was achieved by improving the synthesis ability of 6-methyloctanoic acid compared to the original strain expressing polymyxin heterologously. Additionally, incorporating an anikasin-derived domain into the hybrid nonribosomal peptide synthase of polymyxin increased the B1 ratio in polymyxin B from 57.5% to 62.2%. Through optimization of peptone supply in the fermentation medium and fermentation in a 5.0-L bioreactor, the final polymyxin B titer reached 962.1 mg/L, with a yield of 19.24 mg/g maltodextrin and a productivity of 10.02 mg/(L·h). This study demonstrates a successful approach for enhancing polymyxin B production and increasing the B1 ratio through combinatorial metabolic engineering.

De novo biosynthesis of anthocyanins in Saccharomyces cerevisiae using metabolic pathway synthases from blueberry.

BACKGROUND: Anthocyanins are water-soluble flavonoids in plants, which give plants bright colors and are widely used as food coloring agents, nutrients, and cosmetic additives. There are several limitations for traditional techniques of collecting anthocyanins from plant tissues, including species, origin, season, and technology. The benefits of using engineering microbial production of natural products include ease of use, controllability, and high efficiency. RESULTS: In this study, ten genes encoding enzymes involved in the anthocyanin biosynthetic pathway were successfully cloned from anthocyanin-rich plant materials blueberry fruit and purple round eggplant rind. The Yeast Fab Assembly technology was utilized to construct the transcriptional units of these genes under different promoters. The transcriptional units of PAL and C4H, 4CL and CHS were fused and inserted into Chr. XVI and IV of yeast strain JDY52 respectively using homologous recombination to gain Strain A. The fragments containing the transcriptional units of CHI and F3H, F3'H and DFR were inserted into Chr. III and XVI to gain Strain B1. Strain B2 has the transcriptional units of ANS and 3GT in Chr. IV. Several anthocyanidins, including cyanidin, peonidin, pelargonidin, petunidin, and malvidin, were detected by LC-MS/MS following the predicted outcomes of the de novo biosynthesis of anthocyanins in S. cerevisiae using a multi-strain co-culture technique. CONCLUSIONS: We propose a novel concept for advancing the heterologous de novo anthocyanin biosynthetic pathway, as well as fundamental information and a theoretical framework for the ensuing optimization of the microbial synthesis of anthocyanins.

FOXK2 affects cancer cell response to chemotherapy by promoting nucleotide de novo synthesis.

AIMS: Nucleotide de novo synthesis is essential to cell growth and survival, and its dysregulation leads to cancers and drug resistance. However, how this pathway is dysregulated in cancer has not been well clarified. This study aimed to identify the regulatory mechanisms of nucleotide de novo synthesis and drug resistance. METHODS: By combining the ChIP-Seq data from the Cistrome Data Browser, RNA sequencing (RNA-Seq) and a luciferase-based promoter assay, we identified transcription factor FOXK2 as a regulator of nucleotide de novo synthesis. To explore the biological functions and mechanisms of FOXK2 in cancers, we conducted biochemical and cell biology assays in vitro and in vivo. Finally, we assessed the clinical significance of FOXK2 in hepatocellular carcinoma. RESULTS: FOXK2 directly regulates the expression of nucleotide synthetic genes, promoting tumor growth and cancer cell resistance to chemotherapy. FOXK2 is SUMOylated by PIAS4, which elicits FOXK2 nuclear translocation, binding to the promoter regions and transcription of nucleotide synthetic genes. FOXK2 SUMOylation is repressed by DNA damage, and elevated FOXK2 SUMOylation promotes nucleotide de novo synthesis which causes resistance to 5-FU in hepatocellular carcinoma. Clinically, elevated expression of FOXK2 in hepatocellular carcinoma patients was associated with increased nucleotide synthetic gene expression and correlated with poor prognoses for patients. CONCLUSION: Our findings establish FOXK2 as a novel regulator of nucleotide de novo synthesis, with potentially important implications for cancer etiology and drug resistance.

Facile, Direct, De Novo Synthesis of an Alkyl Phosphoric Acid-Decorated Covalent Organic Framework.

The development and understanding of proton conductors based on phosphoric acid are critical for the field of chemistry, biology, and energy. Covalent organic frameworks (COFs), featuring highly crystalline structures and controllable pore sizes, are suitable for constructing phosphoric acid-based proton conductors. However, because of tedious and intricate synthesis, how to develop COFs based on phosphoric acid remains a substantial challenge. Herein, a side-chain decorated strategy is contributed to construct a phosphoric acid-functionalized, imine-linked COF by de novo synthesis. The phosphoric acid side chains with vigorous motion integrating with 1D nanochannels endow the resulting COF with intrinsic proton conductivity. This work expectantly provides a competitive alternative for producing phosphoric acid-functionalized COFs with high intrinsic proton conductivity.

LASSO Regression with Multiple Imputations for the Selection of Key Variables Affecting the Fatty Acid Profile of Nannochloropsis oculata.

The marine microalga Nannochloropsis oculata has garnered significant interest as a potential source of lipids, both for biofuel and nutrition, containing significant amounts of C16:0, C16:1, and C20:5, n-3 (EPA) fatty acids (FA). Growth parameters such as temperature, pH, light intensity, and nutrient availability play a crucial role in the fatty acid profile of microalgae, with N. oculata being no exception. This study aims to identify key variables for the FA profile of N. oculata grown autotrophically. To that end, the most relevant literature data were gathered and combined with our previous work as well as with novel experimental data, with 121 observations in total. The examined variables were the percentages of C14:0, C16:0, C16:1, C18:1, C18:2, and C20:5, n-3 in total FAs, their respective ratios to C16:0, and the respective content of biomass in those fatty acids in terms of ash free dry weight. Many potential predictor variables were collected, while dummy variables were introduced to account for bias in the measured variables originating from different authors as well as for other parameters. The method of multiple imputations was chosen to handle missing data, with limits based on the literature and model-based estimation, such as using the software PHREEQC and residual modelling for the estimation of pH. To eliminate unimportant predictor variables, LASSO (Least Absolute Shrinkage and Selection Operator) regression analysis with a novel definition of optimal lambda was employed. LASSO regression identified the most relevant predictors while minimizing the risk of overfitting the model. Subsequently, stepwise linear regression with interaction terms was used to further study the effects of the selected predictors. After two rounds of regression, sparse refined models were acquired, and their coefficients were evaluated based on significance. Our analysis confirms well-known effects, such as that of temperature, and it uncovers novel unreported effects of aeration, calcium, magnesium, and manganese. Of special interest is the negative effect of aeration on polyunsaturated fatty acids (PUFAs), which is possibly related to the enzymatic kinetics of fatty acid desaturation under increased oxygen concentration. These findings contribute to the optimization of the fatty acid profile of N. oculata for different purposes, such as production of, high in PUFAs, food or feed, or production of, high in saturated and monounsaturated FA methyl esters (FAME), biofuels.

Research Progress in Improving Photosynthetic Efficiency.

Photosynthesis is the largest mass- and energy-conversion process on Earth, and it is the material basis for almost all biological activities. The efficiency of converting absorbed light energy into energy substances during photosynthesis is very low compared to theoretical values. Based on the importance of photosynthesis, this article summarizes the latest progress in improving photosynthesis efficiency from various perspectives. The main way to improve photosynthetic efficiency is to optimize the light reactions, including increasing light absorption and conversion, accelerating the recovery of non-photochemical quenching, modifying enzymes in the Calvin cycle, introducing carbon concentration mechanisms into C3 plants, rebuilding the photorespiration pathway, de novo synthesis, and changing stomatal conductance. These developments indicate that there is significant room for improvement in photosynthesis, providing support for improving crop yields and mitigating changes in climate conditions.

Review of Eukaryote Cellular Membrane Lipid Composition, with Special Attention to the Fatty Acids.

Biological membranes, primarily composed of lipids, envelop each living cell. The intricate composition and organization of membrane lipids, including the variety of fatty acids they encompass, serve a dynamic role in sustaining cellular structural integrity and functionality. Typically, modifications in lipid composition coincide with consequential alterations in universally significant signaling pathways. Exploring the various fatty acids, which serve as the foundational building blocks of membrane lipids, provides crucial insights into the underlying mechanisms governing a myriad of cellular processes, such as membrane fluidity, protein trafficking, signal transduction, intercellular communication, and the etiology of certain metabolic disorders. Furthermore, comprehending how alterations in the lipid composition, especially concerning the fatty acid profile, either contribute to or prevent the onset of pathological conditions stands as a compelling area of research. Hence, this review aims to meticulously introduce the intricacies of membrane lipids and their constituent fatty acids in a healthy organism, thereby illuminating their remarkable diversity and profound influence on cellular function. Furthermore, this review aspires to highlight some potential therapeutic targets for various pathological conditions that may be ameliorated through dietary fatty acid supplements. The initial section of this review expounds on the eukaryotic biomembranes and their complex lipids. Subsequent sections provide insights into the synthesis, membrane incorporation, and distribution of fatty acids across various fractions of membrane lipids. The last section highlights the functional significance of membrane-associated fatty acids and their innate capacity to shape the various cellular physiological responses.

Nucleotide Imbalance, Provoked by Downregulation of Aspartate Transcarbamoylase Impairs Cold Acclimation in Arabidopsis.

Aspartate transcarbamoylase (ATC) catalyzes the first committed step in pyrimidine de novo synthesis. As shown before, mutants with 80% reduced transcript and protein levels exhibit reduced levels of pyrimidine metabolites and thus nucleotide limitation and imbalance. Consequently, reduced photosynthetic capacity and growth, accompanied by massive transcriptional changes, were observed. Here, we show that nucleotide de novo synthesis was upregulated during cold acclimation of Arabidopsis thaliana (ecotype Columbia, Col-0) plants, but ATC knockdown mutants failed to acclimate to this condition as they did not accumulate neutral sugars and anthocyanins. A global transcriptome analysis revealed that most of the transcriptional changes observed in Col-0 plants upon cold exposure were also evident in ATC knockdown plants. However, several responses observed in cold-treated Col-0 plants could already be detected in knockdown plants when grown under standard conditions, suggesting that these mutants exhibited typical cold responses without prior cold stimulation. We believe that nucleotide signaling is involved in "cold-like priming" and "cold acclimation" in general. The observed transcript levels of genes involved in central carbon metabolism and respiration were an exception to these findings. These were upregulated in the cold but downregulated in warm-grown ATC mutants.

Characterization of mating type on aroma production and metabolic properties wild Kluyveromyces marxianus yeasts.

Kluyveromyces marxianus yeasts represent a valuable industry alternative due to their biotechnological potential to produce aromatic compounds. 2-phenylethanol and 2-phenylethylacetate are significant aromatic compounds widely used in food and cosmetics due to their pleasant odor. Natural obtention of these compounds increases their value, and because of this, bioprocesses such as de novo synthesis has become of great significance. However, the relationship between aromatic compound production and yeast's genetic diversity has yet to be studied. In the present study, the analysis of the genetic diversity in K. marxianus isolated from the natural fermentation of Agave duranguensis for Mezcal elaboration is presented. The results of strains in a haploid and diploid state added to the direct relationship between the mating type locus MAT with metabolic characteristics are studied. Growth rate, assimilate carbohydrates (glucose, lactose, and chicory inulin), and the production of aromatic compounds such as ethyl acetate, isoamyl acetate, isoamyl alcohol, 2-phenylethyl butyrate and phenylethyl propionate and the diversity in terms of the output of 2-phenylethanol and 2-phenylethylacetate by de novo synthesis were determinate, obtaining maximum concentrations of 51.30 and 60.39 mg/L by ITD0049 and ITD 0136 yeasts respectively.

[Anemoside B4 regulates fatty acid metabolism reprogramming in mice with colitis-associated cancer].

The study aimed to investigate the effect of anemoside B4(B4) on fatty acid metabolism in mice with colitis-associated cancer(CAC). The CAC model was established by azoxymethane(AOM)/dextran sodium sulfate(DSS) in mice. Mice were randomly divided into a normal group, a model group, and low-, medium-, and high-dose anemoside B4 groups. After the experiment, the length of the mouse colon and the size of the tumor were measured, and the pathological alterations in the mouse colon were observed using hematoxylin-eosin(HE) staining. The slices of the colon tumor were obtained for spatial metabolome analysis to analyze the distribution of fatty acid metabolism-related substances in the tumor. The mRNA levels of SREBP-1, FAS, ACCα, SCD-1, PPARα, ACOX, UCP-2, and CPT-1 were determined by real-time quantitative PCR(RT-qPCR). The results revealed that the model group showed decreased body weight(P<0.05) and colon length(P<0.001), increased number of tumors, and increased pathological score(P<0.01). Spatial metabolome analysis revealed that the content of fatty acids and their derivatives, carnitine, and phospholipid in the colon tumor was increased. RT-qPCR results indicated that fatty acid de novo synthesis and ß-oxidation-related genes, such as SREBP-1, FASN, ACCα, SCD-1, ACOX, UCP-2, and CPT-1 mRNA expression levels increased considerably(P<0.05, P<0.001). After anemoside B4 administration, the colon length increased(P<0.01), and the number of tumors decreased in the high-dose anemoside B4 group(P<0.05). Additionally, spatial metabolome analysis showed that anemoside B4 could decrease the content of fatty acids and their derivatives, carnitine, and phospholipids in colon tumors. Meanwhile, anemoside B4 could also down-regulate the expression of FASN, ACCα, SCD-1, PPARα, ACOX, UCP-2, and CPT-1 in the colon(P<0.05, P<0.01, P<0.001). The findings of this study show that anemoside B4 may inhibit CAC via regulating fatty acid metabolism reprogramming.

Multilevel metabolic engineering of Bacillus licheniformis for de novo biosynthesis of 2-phenylethanol.

Due to its pleasant rose-like scent, 2-phenylethanol (2-PE) has been widely used in the fields of cosmetics and food. Microbial production of 2-PE offers a natural and sustainable production process. However, the current bioprocesses for de novo production of 2-PE suffer from low titer, yield, and productivity. In this work, a multilevel metabolic engineering strategy was employed for the high-level production of 2-PE. Firstly, the native alcohol dehydrogenase YugJ was identified and characterized for 2-PE production via genome mining and gene function analysis. Subsequently, the redirection of carbon flux into 2-PE biosynthesis by combining optimization of Ehrlich pathway, central metabolic pathway, and phenylpyruvate pathway enabled the production of 2-PE to a titer of 1.81 g/L. Specifically, AroK and AroD were identified as the rate-limiting enzymes of 2-PE production through transcription and metabolite analyses, and overexpression of aroK and aroD efficiently boosted 2-PE synthesis. The precursor competing pathways were blocked by eliminating byproduct formation pathways and modulating the glucose transport system. Under the optimal condition, the engineered strain PE23 produced 6.24 g/L of 2-PE with a yield and productivity of 0.14 g/g glucose and 0.13 g/L/h, respectively, using a complex medium in shake flasks. This work achieves the highest titer, yield, and productivity of 2-PE from glucose via the phenylpyruvate pathway. This study provides a promising platform that might be widely useful for improving the production of aromatic-derived chemicals.

Engineering synthetic microbial consortium for cadaverine biosynthesis from glycerol.

OBJECTIVES: 1,5-pentanediamine (cadaverine) is a C5 platform chemical, also an important raw material for bio-polyamide PA5X. With increasing concerns about the depletion of fossil resources and global environmental protection, cadaverine bio-production has attracted more attentions. RESULTS: Here, a microbial consortium consisting of Corynebacterium glutamicum cgl-FDK and Escherichia coli BL-ABST-Spy was constructed to de novo synthesize cadaverine utilizing glycerol as the sole carbon resource. The glycerol utilization pathway was initially constructed in C. glutamicum cgl-FDK to produce lysine from glycerol. Then, the pyridoxal 5'-phosphate (PLP) biosynthesis pathway and SpyTag/SpyCatcher protein-ligation system for lysine decarboxylase (CadA) and cadaverine-lysine antiporter protein (CadB) were introduced into E. coli BL-ABST-Spy to synthesize cadaverine from lysine. Furthermore, the fermentation conditions of microbial consortium were optimized and the cadaverine production reached 9.3 g/L with glycerol as the sole carbon source. CONCLUSIONS: This work provides a promising strategy for efficiently producing cadaverine from glycerol with an artificial microbial consortium.

The Association of Cholesterol Uptake and Synthesis with Histology and Genotype in Cortisol-Producing Adenoma (CPA).

Cortisol-producing adenoma (CPA) is composed of clear and compact cells. Clear cells are lipid abundant, and compact ones lipid poor but associated with higher production of steroid hormones. PRKACA mutation (PRKACA mt) in CPA patients was reported to be associated with more pronounced clinical manifestation of Cushing's syndrome. In this study, we examined the association of histological features and genotypes with cholesterol uptake receptors and synthetic enzymes in 40 CPA cases, and with the quantitative results obtained by gas chromatography-mass spectrometry (GC-MS) analysis in 33 cases to explore their biological and clinical significance. Both cholesterol uptake receptors and synthetic enzymes were more abundant in compact cells. GC-MS analysis demonstrated that the percentage of compact cells was inversely correlated with the concentrations of cholesterol and cholesterol esters, and positively with the activity of cholesterol biosynthesis from cholesterol esters. In addition, hormone-sensitive lipase (HSL), which catalyzes cholesterol biosynthesis from cholesterol esters, tended to be more abundant in compact cells of PRKACA mt CPAs. These results demonstrated that both cholesterol uptake and biosynthesis were more pronounced in compact cells in CPA. In addition, more pronounced HSL expression in compact cells of PRKACA mt CPA could contribute to their more pronounced clinical manifestation.

Advances in Atroposelectively De Novo Synthesis of Axially Chiral Heterobiaryl Scaffolds.

Axially chiral heterobiaryl frameworks are privileged structures in many natural products, pharmaceutically active molecules, and chiral ligands. Therefore, a variety of approaches for constructing these skeletons have been developed. Among them, de novo synthesis, due to its highly convergent and superior atom economy, serves as a promising strategy to access these challenging scaffolds including C-N, C-C, and N-N chiral axes. So far, several elegant reviews on the synthesis of axially chiral heterobiaryl skeletons have been disclosed, however, atroposelective construction of the heterobiaryl subunits by de novo synthesis was rarely covered. Herein, we summarized the recent advances in the catalytic asymmetric synthesis of the axially chiral heterobiaryl scaffold via de novo synthetic strategies. The related mechanism, scope, and applications were also included.

Sequential Metal Catalysis towards 7-Oxostaurosporine and Its Non-Natural Septanose Analogue.

We report sequential metal catalysis towards indolocarbazole glycosides. The signature event is highlighted by i) Pd0 -catalyzed addition of indolocarbazole to alkoxyallene combined with ring-closing-metathesis; ii) Ru-catalyzed chemoselective olefin migration; iii) PdII -catalyzed oxidative cyclization to build the bicyclic core structure of the target compounds. This approach gave access to both natural pyranose- and non-natural septanose glycosides. A short formal synthesis of 7-oxostaurosporine was achieved via this strategy.

Total Synthesis of Starfish Cyclic Steroid Glycosides.

Starfishes have evolved with a special type of secondary metabolites, namely starfish saponins, to ward off various predators and parasites; among them, the starfish cyclic steroid glycosides stand out structurally, featuring a unique 16-membered ring formed by bridging the steroidal C3 and C6 with a trisaccharide. The rigid cyclic scaffold and the congested and vulnerable steroid-sugar etherate linkage present an unprecedented synthetic challenge. Here we report a collective total synthesis of the major starfish cyclic steroid glycosides, namely luzonicosides A (1) and D (2) and sepositoside A (3), with an innovative approach, which entails a de novo construction of the ether-linked hexopyranosyl units, use of olefinic pyranoses as sugar precursors, and a decisive ring-closing glycosylation under the mild gold(I)-catalyzed conditions.

Cholesterol homeostasis in the vertebrate retina: biology and pathobiology.

Cholesterol is a quantitatively and biologically significant constituent of all mammalian cell membrane, including those that comprise the retina. Retinal cholesterol homeostasis entails the interplay between de novo synthesis, uptake, intraretinal sterol transport, metabolism, and efflux. Defects in these complex processes are associated with several congenital and age-related disorders of the visual system. Herein, we provide an overview of the following topics: (a) cholesterol synthesis in the neural retina; (b) lipoprotein uptake and intraretinal sterol transport in the neural retina and the retinal pigment epithelium (RPE); (c) cholesterol efflux from the neural retina and the RPE; and (d) biology and pathobiology of defects in sterol synthesis and sterol oxidation in the neural retina and the RPE. We focus, in particular, on studies involving animal models of monogenic disorders pertinent to the above topics, as well as in vitro models using biochemical, metabolic, and omic approaches. We also identify current knowledge gaps and opportunities in the field that beg further research in this topic area.

Synthesis of Piperidine Nucleosides as Conformationally Restricted Immucillin Mimics.

The de novo synthesis of piperidine nucleosides from our homologating agent 5,6-dihydro-1,4-dithiin is herein reported. The structure and conformation of nucleosides were conceived to faithfully resemble the well-known nucleoside drugs Immucillins H and A in their bioactive conformation. NMR analysis of the synthesized compounds confirmed that they adopt an iminosugar conformation bearing the nucleobases and the hydroxyl groups in the appropriate orientation.

Increasing Sphingolipid Synthesis Alleviates Airway Hyperreactivity.

Impaired sphingolipid synthesis is linked genetically to childhood asthma and functionally to airway hyperreactivity (AHR). The objective was to investigate whether sphingolipid synthesis could be a target for asthma therapeutics. The effects of GlyH-101 and fenretinide via modulation of de novo sphingolipid synthesis on AHR was evaluated in mice deficient in SPT (serine palmitoyl-CoA transferase), the rate-limiting enzyme of sphingolipid synthesis. The drugs were also used directly in human airway smooth-muscle and epithelial cells to evaluate changes in de novo sphingolipid metabolites and calcium release. GlyH-101 and fenretinide increased sphinganine and dihydroceramides (de novo sphingolipid metabolites) in lung epithelial and airway smooth-muscle cells, decreased the intracellular calcium concentration in airway smooth-muscle cells, and decreased agonist-induced contraction in proximal and peripheral airways. GlyH-101 also decreased AHR in SPT-deficient mice in vivo. This study identifies the manipulation of sphingolipid synthesis as a novel metabolic therapeutic strategy to alleviate AHR.

Exploiting the Diversity of Saccharomycotina Yeasts To Engineer Biotin-Independent Growth of Saccharomyces cerevisiae.

Biotin, an important cofactor for carboxylases, is essential for all kingdoms of life. Since native biotin synthesis does not always suffice for fast growth and product formation, microbial cultivation in research and industry often requires supplementation of biotin. De novo biotin biosynthesis in yeasts is not fully understood, which hinders attempts to optimize the pathway in these industrially relevant microorganisms. Previous work based on laboratory evolution of Saccharomyces cerevisiae for biotin prototrophy identified Bio1, whose catalytic function remains unresolved, as a bottleneck in biotin synthesis. This study aimed at eliminating this bottleneck in the S. cerevisiae laboratory strain CEN.PK113-7D. A screening of 35 Saccharomycotina yeasts identified six species that grew fast without biotin supplementation. Overexpression of the S. cerevisiaeBIO1 (ScBIO1) ortholog isolated from one of these biotin prototrophs, Cyberlindnera fabianii, enabled fast growth of strain CEN.PK113-7D in biotin-free medium. Similar results were obtained by single overexpression of C. fabianii BIO1 (CfBIO1) in other laboratory and industrial S. cerevisiae strains. However, biotin prototrophy was restricted to aerobic conditions, probably reflecting the involvement of oxygen in the reaction catalyzed by the putative oxidoreductase CfBio1. In aerobic cultures on biotin-free medium, S. cerevisiae strains expressing CfBio1 showed a decreased susceptibility to contamination by biotin-auxotrophic S. cerevisiae This study illustrates how the vast Saccharomycotina genomic resources may be used to improve physiological characteristics of industrially relevant S. cerevisiaeIMPORTANCE The reported metabolic engineering strategy to enable optimal growth in the absence of biotin is of direct relevance for large-scale industrial applications of S. cerevisiae Important benefits of biotin prototrophy include cost reduction during the preparation of chemically defined industrial growth media as well as a lower susceptibility of biotin-prototrophic strains to contamination by auxotrophic microorganisms. The observed oxygen dependency of biotin synthesis by the engineered strains is relevant for further studies on the elucidation of fungal biotin biosynthesis pathways.