Recombinant lactate-assimilating cyanobacteria reduce high-concentration culture-associated cytotoxicity in mammalian cells.

In the fields of cultured meat, biopharmaceuticals, cell therapy, and tissue engineering, large numbers of mammalian cells are required; thus, highly-concentrated cell cultures are widely adopted. In general, such cultures can lead to cell damage caused by waste product accumulation and nutritional inadequacy. In this study, a novel co-culture system where the recombinant lactate-assimilating cyanobacterial strain, KC0110, derived from euryhaline Picosynechococcus sp. PCC 7002, and mammalian muscle cells cultured across porous membranes been developed. By using the KC0110 strain, the amount of ammonium and lactate excreted from C2C12 mouse muscle cells into the culture significantly decreased. Importantly, pyruvate and some amino acids, including pyruvate-derived amino acids, also increased significantly compared to those in monoculture of C2C12 cells. It is believed that the organic acids secreted by the KC0110 strain enhance the growth of mammalian cells, leading to a reduction in high-concentration culture-induced mammalian cell damage [lactate dehydrogenase (LDH) release] through cyanobacterial co-culture. These results show that, through co-cultivation with cyanobacteria, it is possible to culture mammalian cells, alleviating cell damage, even in highly-concentrated cultures. This study demonstrated an in vitro "symbiotic circular system" that can interchange metabolites produced by phototrophs and mammalian cells.

Pioneering precision in markerless strain development for Synechococcus sp. PCC 7002.

Marine cyanobacteria such as Picosynechococcus sp. (formerly called Synechococcus sp.) PCC 7002 are promising chassis for photosynthetic production of commodity chemicals with low environmental burdens. Genetic engineering of cyanobacteria conventionally employs antibiotic resistance markers. However, limited availability of antibiotic-resistant markers is a problem for highly multigenic strain engineering. Although several markerless genetic manipulation methods have been developed for PCC 7002, they often lack versatility due to the requirement of gene disruption in the host strain. To achieve markerless transformation in Synechococcus sp. with no requirements for the host strain, this study developed a method in which temporarily introduces a mutated phenylalanyl-tRNA synthetase gene (pheS) into the genome for counter selection. Amino acid substitutions in the PheS that cause high susceptibility of PCC 7002 to the phenylalanine analog p-chlorophenylalanine were examined, and the combination of T261A and A303G was determined as the most suitable mutation. The mutated PheS-based selection was utilized for the markerless knockout of the nblA gene in PCC 7002. In addition, the genetic construct containing the lldD and lldP genes from Escherichia coli was introduced into the ldhA gene site using the counter selection strategy, resulting in a markerless recombinant strain. The repeatability of this method was demonstrated by the double markerless knockin recombinant strain, suggesting it will be a powerful tool for multigenic strain engineering of cyanobacteria.

Minimized dark consumption of Calvin cycle intermediates facilitates the initiation of photosynthesis in Synechocystis sp. PCC 6803.

Cyanobacteria intricately regulate their metabolic pathways during the diurnal cycle to ensure survival and growth. Under dark conditions, the breakdown of glycogen, an energy reserve, in these organisms replenishes Calvin cycle intermediates, especially downstream glycolytic metabolites, which are necessary for photosynthesis initiation upon light irradiation. However, it remains unclear how the accumulation of these intermediates is maintained in the dark despite limited glycogen availability. Therefore, in this study, we investigated the regulation of downstream glycolytic metabolites of the Calvin cycle under dark and light treatment using Synechocystis sp. PCC 6803. Our results showed that during the dark period, low pyruvate kinase (Pyk) activity ensured metabolite accumulation, while endogenous Pyk overexpression significantly lowered the accumulation of glycolytic intermediates. Remarkably, wild type Synechocystis maintained oxygen evolution ability throughout dark treatment for over 2 d, while Pyk overexpression resulted in decreased oxygen evolution after 16 h of dark treatment. These results indicated that limiting Pyk activity via darkness treatment facilitates photosynthetic initiation by maintaining glycolytic intermediates. Similarly, phosphoenolpyruvate carboxylase (PepC) overexpression decreased oxygen evolution under dark treatment; however, its effect was lower than that of Pyk. Further, we noted that as PepC overexpression decreased the levels of glycolytic intermediates in the dark, sugar phosphates in the Calvin-Benson-Bassham (CBB) cycle showed high accumulation, suggesting that sugar phosphates play important roles in supporting photosynthesis initiation. Therefore, our study highlights the importance of controlling the metabolic pathways through which glycolytic and CBB cycle intermediates are consumed (defined as cataplerosis of CBB cycle) to ensure stable photosynthesis.

One-pot synthesis of cellobiose from sucrose using sucrose phosphorylase and cellobiose phosphorylase co-displaying Pichia pastoris as a reusable whole-cell biocatalyst.

Cellobiose has received increasing attention in various industrial sectors, ranging from food and feed to cosmetics. The development of large-scale cellobiose applications requires a cost-effective production technology as currently used methods based on cellulose hydrolysis are costly. Here, a one-pot synthesis of cellobiose from sucrose was conducted using a recombinant Pichia pastoris strain as a reusable whole-cell biocatalyst. Thermophilic sucrose phosphorylase from Bifidobacterium longum (BlSP) and cellobiose phosphorylase from Clostridium stercorarium (CsCBP) were co-displayed on the cell surface of P. pastoris via a glycosylphosphatidylinositol-anchoring system. Cells of the BlSP and CsCBP co-displaying P. pastoris strain were used as whole-cell biocatalysts to convert sucrose to cellobiose with commercial thermophilic xylose isomerase. Cellobiose productivity significantly improved with yeast cells grown on glycerol compared to glucose-grown cells. In one-pot bioconversion using glycerol-grown yeast cells, approximately 81.2 g/L of cellobiose was produced from 100 g/L of sucrose, corresponding to 81.2% of the theoretical maximum yield, within 24 h at 60 °C. Moreover, recombinant yeast cells maintained a cellobiose titer > 80 g/L, even after three consecutive cell-recycling one-pot bioconversion cycles. These results indicated that one-pot bioconversion using yeast cells displaying two phosphorylases as whole-cell catalysts is a promising approach for cost-effective cellobiose production.

A serum-free culture medium production system by co-culture combining growth factor-secreting cells and L-lactate-assimilating cyanobacteria for sustainable cultured meat production.

Large-scale production of cultured meat requires bulk culture medium containing growth-promoting proteins from animal serum. However, animal serum for mammalian cell culture is associated with high costs, ethical concerns, and contamination risks. Owing to its growth factor content, conditioned medium from rat liver epithelial RL34 cells can replace animal serum for myoblast proliferation. More seeded cells and longer culture periods are thought to yield higher growth factor levels, resulting in more effective muscle cell proliferation. However, RL34 cells can deplete nutrients and release harmful metabolites into the culture medium over time, potentially causing growth inhibition and apoptosis. This issue highlights the need for waste clearance during condition medium production. To address this issue, we introduced a lactate permease gene (lldP) and an L-lactate-to-pyruvate conversion enzyme gene (lldD) to generate a recombinant L-lactate-assimilating cyanobacterium Synechococcus sp. KC0110 strain. Transwell co-culture of this strain with RL34 cells exhibited a marked reduction in the levels of harmful metabolites, lactate and ammonium, while maintaining higher concentrations of glucose, pyruvate, and pyruvate-derived amino acids than those seen with RL34 cell monocultures. The co-culture medium supported myoblast proliferation without medium dilution or additional nutrients, which was attributed to the waste clearance and nutrient replenishment effects of the KC0110 strain. This culture system holds potential for the production of low-cost, and animal-free cultured meat.

Engineering Escherichia coli for efficient glutathione production.

Glutathione is a tripeptide of excellent value in the pharmaceutical, food, and cosmetic industries that is currently produced during yeast fermentation. In this case, glutathione accumulates intracellularly, which hinders high production. Here, we engineered Escherichia coli for the efficient production of glutathione. A total of 4.3 g/L glutathione was produced by overexpressing gshA and gshB, which encode cysteine glutamate ligase and glutathione synthetase, respectively, and most of the glutathione was excreted into the culture medium. Further improvements were achieved by inhibiting degradation (Δggt and ΔpepT); deleting gor (Δgor), which encodes glutathione oxide reductase; attenuating glutathione uptake (ΔyliABCD); and enhancing cysteine production (PompF-cysE). The engineered strain KG06 produced 19.6 g/L glutathione after 48 h of fed-batch fermentation with continuous addition of ammonium sulfate as the sulfur source. We also found that continuous feeding of glycine had a crucial role for effective glutathione production. The results of metabolic flux and metabolomic analyses suggested that the conversion of O-acetylserine to cysteine is the rate-limiting step in glutathione production by KG06. The use of sodium thiosulfate largely overcame this limitation, increasing the glutathione titer to 22.0 g/L, which is, to our knowledge, the highest titer reported to date in the literature. This study is the first report of glutathione fermentation without adding cysteine in E. coli. Our findings provide a great potential of E. coli fermentation process for the industrial production of glutathione.

High-throughput evaluation of hemolytic activity through precise measurement of colony and hemolytic zone sizes of engineered Bacillus subtilis on blood agar.

Biosurfactants have remarkable characteristics, such as environmental friendliness, high safety, and excellent biodegradability. Surfactin is one of the best-known biosurfactants produced by Bacillus subtilis. Because the biosynthetic pathways of biosurfactants, such as surfactin, are complex, mutagenesis is a useful alternative to typical metabolic engineering approaches for developing high-yield strains. Therefore, there is a need for high-throughput and accurate screening methods for high-yield strains derived from mutant libraries. The blood agar lysis method, which takes advantage of the hemolytic activity of biosurfactants, is one way of determining their concentration. This method includes inoculating microbial cells onto blood-containing agar plates, and biosurfactant production is assessed based on the size of the hemolytic zone formed around each colony. Challenges with the blood agar lysis method include low experimental reproducibility and a lack of established protocols for high-throughput screening. Therefore, in this study, we investigated the effects of the inoculation procedure and media composition on the formation of hemolytic zones. We also developed a workflow to evaluate the number of colonies using robotics. The results revealed that by arranging colonies at appropriate intervals and measuring the areas of colonies and hemolytic rings using image analysis software, it was possible to accurately compare the hemolytic activity among several colonies. Although the use of the blood agar lysis method for screening is limited to surfactants exhibiting hemolytic activity, it is believed that by considering the insights gained from this study, it can contribute to the accurate screening of strains with high productivity.

Integrated pathway mining and selection of an artificial CYP79-mediated bypass to improve benzylisoquinoline alkaloid biosynthesis.

BACKGROUND: Computational mining of useful enzymes and biosynthesis pathways is a powerful strategy for metabolic engineering. Through systematic exploration of all conceivable combinations of enzyme reactions, including both known compounds and those inferred from the chemical structures of established reactions, we can uncover previously undiscovered enzymatic processes. The application of the novel alternative pathways enables us to improve microbial bioproduction by bypassing or reinforcing metabolic bottlenecks. Benzylisoquinoline alkaloids (BIAs) are a diverse group of plant-derived compounds with important pharmaceutical properties. BIA biosynthesis has developed into a prime example of metabolic engineering and microbial bioproduction. The early bottleneck of BIA production in Escherichia coli consists of 3,4-dihydroxyphenylacetaldehyde (DHPAA) production and conversion to tetrahydropapaveroline (THP). Previous studies have selected monoamine oxidase (MAO) and DHPAA synthase (DHPAAS) to produce DHPAA from dopamine and oxygen; however, both of these enzymes produce toxic hydrogen peroxide as a byproduct. RESULTS: In the current study, in silico pathway design is applied to relieve the bottleneck of DHPAA production in the synthetic BIA pathway. Specifically, the cytochrome P450 enzyme, tyrosine N-monooxygenase (CYP79), is identified to bypass the established MAO- and DHPAAS-mediated pathways in an alternative arylacetaldoxime route to DHPAA with a peroxide-independent mechanism. The application of this pathway is proposed to result in less formation of toxic byproducts, leading to improved production of reticuline (up to 60 mg/L at the flask scale) when compared with that from the conventional MAO pathway. CONCLUSIONS: This study showed improved reticuline production using the bypass pathway predicted by the M-path computational platform. Reticuline production in E. coli exceeded that of the conventional MAO-mediated pathway. The study provides a clear example of the integration of pathway mining and enzyme design in creating artificial metabolic pathways and suggests further potential applications of this strategy in metabolic engineering.

Mixotrophic culture enhances fucoxanthin production in the haptophyte Pavlova gyrans.

Fucoxanthin is a versatile substance in the food and pharmaceutical industries owing to its excellent antioxidant and anti-obesity properties. Several microalgae, including the haptophyte Pavlova spp., can produce fucoxanthin and are potential industrial fucoxanthin producers, as they lack rigid cell walls, which facilitates fucoxanthin extraction. However, the commercial application of Pavlova spp. is limited owing to insufficient biomass production. In this study, we aimed to develop a mixotrophic cultivation method to increase biomass and fucoxanthin production in Pavlova gyrans OPMS 30543X. The effects of culturing OPMS 30543X with different organic carbon sources, glycerol concentrations, mixed-nutrient conditions, and light intensities on the consumption of organic carbon sources, biomass production, and fucoxanthin accumulation were analyzed. Several organic carbon sources, such as glycerol, glucose, sucrose, and acetate, were examined, revealing that glycerol was well-consumed by the microalgae. Biomass and fucoxanthin production by OPMS 30543X increased in the presence of 10 mM glycerol compared to that observed without glycerol. Metabolomic analysis revealed higher levels of the metabolites related to the glycolytic, Calvin-Benson-Bassham, and tricarboxylic acid cycles under mixotrophic conditions than under autotrophic conditions. Cultures grown under mixotrophic conditions with a light intensity of 100 µmol photons m-2 s-1 produced more fucoxanthin than autotrophic cultures. Notably, the amount of fucoxanthin produced (18.9 mg/L) was the highest reported thus far for Pavlova species. In conclusion, the use of mixotrophic culture is a promising strategy for increasing fucoxanthin production in Pavlova species. KEY POINTS: • Glycerol enhances biomass and fucoxanthin production in Pavlova gyrans • Metabolite levels increase under mixotrophic conditions • Mixotrophic conditions and medium-light intensity are appropriate for P. gyrans.

Glycogen deficiency enhances carbon partitioning into glutamate for an alternative extracellular metabolic sink in cyanobacteria.

Glycogen serves as a metabolic sink in cyanobacteria. Glycogen deficiency causes the extracellular release of distinctive metabolites such as pyruvate and 2-oxoglutarate upon nitrogen depletion; however, the mechanism has not been fully elucidated. This study aimed to elucidate the mechanism of carbon partitioning in glycogen-deficient cyanobacteria. Extracellular and intracellular metabolites in a glycogen-deficient ΔglgC mutant of Synechococcus elongatus PCC 7942 were comprehensively analyzed. In the presence of a nitrogen source, the ΔglgC mutant released extracellular glutamate rather than pyruvate and 2-oxoglutarate, whereas its intracellular glutamate level was lower than that in the wild-type strain. The de novo synthesis of glutamate increased in the ΔglgC mutant, suggesting that glycogen deficiency enhanced carbon partitioning into glutamate and extracellular excretion through an unidentified transport system. This study proposes a model in which glutamate serves as the prime extracellular metabolic sink alternative to glycogen when nitrogen is available.

The potency of mitochondria enlargement for mitochondria-mediated terpenoid production in yeast.

Terpenoids are widely used in the food, beverage, cosmetics, and pharmaceutical industries. Microorganisms have been extensively studied for terpenoid production. In yeast, the introduction of the mevalonate (MVA) pathway in organelles in addition to the augmentation of its own MVA pathway have been challenging. Introduction of the MVA pathway into mitochondria is considered a promising approach for terpenoid production because acetyl-CoA, the starting molecule of the MVA pathway, is abundant in mitochondria. However, mitochondria comprise only a small percentage of the entire cell. Therefore, we hypothesized that increasing the total mitochondrial volume per cell would increase terpenoid production. First, we ascertained that the amounts of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the final molecules of the MVA pathway, were 15-fold higher of the strain expressing the MVA pathway in mitochondria than in the wild-type yeast strain. Second, we found that different deletion mutants induced different mitochondrial volumes by measuring the mitochondrial volume in various deletion mutants affecting mitochondrial morphology; for example,Δmdm32 increased mitochondrial volume, and Δfzo1 decreased it. Finally, the effects of mitochondrial volume on amounts of IPP/DMAPP and terpenoids (squalene or ß-carotene) were investigated using mutants harboring large or small mitochondria expressing the MVA pathway in mitochondria. Amounts of IPP/DMAPP and terpenoids (squalene or ß-carotene) increased when the mitochondrial volume expanded. Introducing the MVA pathway into mitochondria for terpenoid production in yeast may become more attractive by enlarging the mitochondrial volume. KEY POINTS: • IPP/DMAPP content increased in the strain expressing the MVA pathway in mitochondria • IPP/DMAPP and terpenoid contents are positively correlated with mitochondrial volume • Enlarging the mitochondria may improve mitochondria-mediated terpenoid production.

Comprehensive metabolic profiling of Geotrichum candidum and comparison with Saccharomyces cerevisiae.

Geotrichum candidum is a dimorphic yeast used in cheese processing. To our knowledge, no major metabolites have been identified to date in G. candidum except for some amino acid and fatty acid metabolites. This has limited research on the commercial use of G. candidum. In this study, we aimed to analyze temporal changes in the intra- and extra-cellular metabolites of G. candidum and Saccharomyces cerevisiae cultured in YM medium as reference. As a result of metabolite analysis, it was observed that G. candidum tends to accumulate　pentose phosphate pathway compounds, which are involved in nucleic acid synthesis, after 48 h of cultivation when compared to S. cerevisiae. In addition, G. candidum accumulated higher amounts of the antioxidant glutathione in the medium than did S. cerevisiae. In addition, G. candidum accumulated large amounts of B vitamins such as pantothenic acid and nicotinic acid in the medium. Finally, we examined the potential of G. candidum as a host for the production of useful compounds such as pantothenic acid. When cultured in medium supplemented with the pantothenic acid precursor ß-alanine, G. candidum produced 12-fold higher amounts of pantothenic acid (30 µM) than that by S. cerevisiae. This study indicates that G. candidum accumulates various useful compounds that are dissimilar to those produced by S. cerevisiae. Furthermore, G. candidum has the potential to produce useful chemicals under appropriate culture conditions.

High-level phenol bioproduction by engineered Pichia pastoris in glycerol fed-batch fermentation using an efficient pertraction system.

This study aimed to establish a high-level phenol bioproduction system from glycerol through metabolic engineering of the yeast Pichia pastoris (Komagataella phaffii). Introducing tyrosine phenol-lyase to P. pastoris led to a production of 59 mg/L of phenol in flask culture. By employing a strain of P. pastoris that overproduces tyrosine-a precursor to phenol-we achieved a phenol production of 1052 mg/L in glycerol fed-batch fermentation. However, phenol concentrations exceeding 1000 mg/L inhibited P. pastoris growth. A phenol pertraction system utilizing a hollow fiber membrane contactor and tributyrin as the organic solvent was developed to reduce phenol concentration in the culture medium. Integrating this system with glycerol fed-batch fermentation resulted in a 214 % increase in phenol titer (3304 mg/L) compared to glycerol fed-batch fermentation alone. These approaches offer a significant framework for the microbial production of chemicals and materials that are highly toxic to microorganisms.

Beneficial effect of optimizing the expression balance of the mevalonate pathway introduced into the mitochondria on terpenoid production in Saccharomyces cerevisiae.

Terpenoids are used in various industries, and Saccharomyces cerevisiae is a promising microorganism for terpenoid production. Introducing the mevalonate (MVA) pathway into the mitochondria of a strain with an augmented inherent cytosolic MVA pathway increased terpenoid production but also led to the accumulation of toxic pyrophosphate intermediates that negatively affected terpenoid production. We first engineered the inherent MVA pathway in the cytosol and then introduced the MVA pathway into the mitochondria using several promoter combinations, considering the toxicity of pyrophosphate intermediates. However, the highest titer, 183 mg/L, tends to be only 5% higher than that of the strain that only augmented the inherent MVA pathway (SYCM1; 174 mg/L). Next, we hypothesized that, in addition to the toxicity of pyrophosphate, other compounds in the MVA pathway could affect the squalene titer. Thus, we constructed a combinatorial strain library expressing MVA pathway enzymes in the mitochondria with various promoter combinations. The highest squalene titer (230 mg/L) was 32% higher than that of SYCM1. The promoter set revealed that mitigation of mono- and pyrophosphate compound accumulation was important for mitochondrial usage. This study demonstrated that a combinatorial strain library is useful for discovering the optimal gene expression balance in engineering yeast.

Metabolomics-based development of bioproduction processes toward industrial-scale production.

Microbial biomanufacturing offers a promising, environment-friendly platform for next-generation chemical production. However, its limited industrial implementation is attributed to the slow production rates of target compounds and the time-intensive engineering of high-yield strains. This review highlights how metabolomics expedites bioproduction development, as demonstrated through case studies of its integration into microbial strain engineering, culture optimization, and model construction. The Design-Build-Test-Learn (DBTL) cycle serves as a standard workflow for strain engineering. Process development, including the optimization of culture conditions and scale-up, is crucial for industrial production. In silico models facilitate the development of strains and processes. Metabolomics is a powerful driver of the DBTL framework, process development, and model construction.

The chromatin remodeler Ino80 regulates yeast stress tolerance and cell metabolism through modulating nitrogen catabolite repression.

Chromatin remodelers are important in maintaining the dynamic chromatin state in eukaryotic cells, which is essential for epigenetic regulation. Among the remodelers, the multi-subunits complex INO80 plays crucial roles in transcriptional regulation. However, current knowledge of chromatin regulation of the core subunit Ino80 on stress adaptation remains mysterious. Here we revealed that overexpressing the chromatin remodeler Ino80 elevated tolerance to multiple stresses in budding yeast Saccharomyces cerevisiae. Analyses of differential chromatin accessibility and global transcription levels revealed an enrichment of genes involved in NCR (nitrogen catabolite repression) under acetic acid stress. We demonstrated that Ino80 overexpression reduced the histone H3 occupancy in the promoter region of the glutamate dehydrogenase gene GDH2 and the allantoinase gene DAL1. Consistently, the decreased occupancy of nucleosome was revealed in the Ino80-inactivation mutant. Further analyses showed that Ino80 was recruited to the specific DNA locus in the promoter region of GDH2. Consistently, Ino80 overexpression facilitated the utilization of non-preferred nitrogen source to enhance ethanol yield under prolonged acetic acid stress. These results demonstrate that Ino80 plays a crucial role in coordinating carbon and nitrogen metabolism during stress adaptation.

ppGpp accumulation reduces the expression of the global nitrogen homeostasis-modulating NtcA regulon by affecting 2-oxoglutarate levels.

The cyanobacterium Synechococcus elongatus PCC 7942 accumulates alarmone guanosine tetraphosphate (ppGpp) under stress conditions, such as darkness. A previous study observed that artificial ppGpp accumulation under photosynthetic conditions led to the downregulation of genes involved in the nitrogen assimilation system, which is activated by the global nitrogen regulator NtcA, suggesting that ppGpp regulates NtcA activity. However, the details of this mechanism have not been elucidated. Here, we investigate the metabolic responses associated with ppGpp accumulation by heterologous expression of the ppGpp synthetase RelQ. The pool size of 2-oxoglutarate (2-OG), which activates NtcA, is significantly decreased upon ppGpp accumulation. De novo 13C-labeled CO2 assimilation into the Calvin-Benson-Bassham cycle and glycolytic intermediates continues irrespective of ppGpp accumulation, whereas the labeling of 2-OG is significantly decreased under ppGpp accumulation. The low 2-OG levels in the RelQ overexpression cells could be because of the inhibition of metabolic enzymes, including aconitase, which are responsible for 2-OG biosynthesis. We propose a metabolic rearrangement by ppGpp accumulation, which negatively regulates 2-OG levels to maintain carbon and nitrogen balance.

Every road leads to Rome: diverse biosynthetic regulation of plant cell wall-degrading enzymes in filamentous fungi Penicillium oxalicum and Trichoderma reesei.

Cellulases and xylanases are plant cell wall-degrading enzymes (CWDEs) that are critical to sustainable bioproduction based on renewable lignocellulosic biomass to reduce carbon dioxide emission. Currently, these enzymes are mainly produced from filamentous fungi, especially Trichoderma reesei and Penicillium oxalicum. However, an in-depth comparison of these two producers has not been performed. Although both P. oxalicum and T. reesei harbor CWDE systems, they exhibit distinct features regulating the production of these enzymes, mainly through different transcriptional regulatory networks. This review presents the strikingly different modes of genome-wide regulation of cellulase and xylanase biosynthesis in P. oxalicum and T. reesei, including sugar transporters, signal transduction cascades, transcription factors, chromatin remodeling, and three-dimensional organization of chromosomes. In addition, different molecular breeding approaches employed, based on the understanding of the regulatory networks, are summarized. This review highlights the existence of very different regulatory modes leading to the efficient regulation of CWDE production in filamentous fungi, akin to the adage that "every road leads to Rome." An understanding of this divergence may help further improvements in fungal enzyme production through the metabolic engineering and synthetic biology of certain fungal species.

The Carbon Flow Shifts from Primary to Secondary Metabolism during Xylem Vessel Cell Differentiation in Arabidopsis thaliana.

Xylem vessel cell differentiation is characterized by the deposition of a secondary cell wall (SCW) containing cellulose, hemicellulose and lignin. VASCULAR-RELATED NAC-DOMAIN7 (VND7), a plant-specific NAC (NAM, ATAF1/2, and CUC2) transcription factor, is a master regulator of xylem vessel cell differentiation in Arabidopsis (Arabidopsis thaliana). Previous metabolome analysis using the VND7-inducible system in tobacco BY-2 cells successfully revealed significant quantitative changes in primary metabolites during xylem vessel cell differentiation. However, the flow of primary metabolites is not yet well understood. Here, we performed a metabolomic analysis of VND7-inducible Arabidopsis T87 suspension cells. Capillary electrophoresis-time-of-flight mass spectrometry quantified 57 metabolites, and subsequent data analysis highlighted active changes in the levels of UDP-glucose and phenylalanine, which are building blocks of cellulose and lignin, respectively. In a metabolic flow analysis using stable carbon 13 (13C) isotope, the 13C-labeling ratio specifically increased in 3-phosphoglycerate after 12 h of VND7 induction, followed by an increase in shikimate after 24 h of induction, while the inflow of 13C into lactate from pyruvate was significantly inhibited, indicating an active shift of carbon flow from glycolysis to the shikimate pathway during xylem vessel cell differentiation. In support of this notion, most glycolytic genes involved in the downstream of glyceraldehyde 3-phosphate were downregulated following the induction of xylem vessel cell differentiation, whereas genes for the shikimate pathway and phenylalanine biosynthesis were upregulated. These findings provide evidence for the active shift of carbon flow from primary metabolic pathways to the SCW polymer biosynthetic pathway at specific points during xylem vessel cell differentiation.