Rewiring metabolic flux to simultaneously improve malate production and eliminate by-product succinate accumulation by Myceliophthora thermophila.

Although a high titre of malic acid is achieved by filamentous fungi, by-product succinic acid accumulation leads to a low yield of malic acid and is unfavourable for downstream processing. Herein, we conducted a series of metabolic rewiring strategies in a previously constructed Myceliophthora thermophila to successfully improve malate production and abolish succinic acid accumulation. First, a pyruvate carboxylase CgPYC variant with increased activity was obtained using a high-throughput system and introduced to improve malic acid synthesis. Subsequently, shifting metabolic flux to malate synthesis from mitochondrial metabolism by deleing mitochondrial carriers of pyruvate and malate, led to a 53.7% reduction in succinic acid accumulation. The acceleration of importing cytosolic succinic acid into the mitochondria for consumption further decreased succinic acid formation by 53.3%, to 2.12 g/L. Finally, the importer of succinic acid was discovered and used to eliminate by-product accumulation. In total, malic acid production was increased by 26.5%, relative to the start strain JG424, to 85.23 g/L and 89.02 g/L on glucose and Avicel, respectively, in the flasks. In a 5-L fermenter, the titre of malic acid reached 182.7 g/L using glucose and 115.8 g/L using raw corncob, without any by-product accumulation. This study would accelerate the industrial production of biobased malic acid from renewable plant biomass.

Fungal carboxylate transporters: recent manipulations and applications.

Carboxylic acids containing acidic groups with additional keto/hydroxyl-groups or unsaturated bond have displayed great applicability in the food, agricultural, cosmetic, textile, and pharmaceutical industries. The traditional approach for carboxylate production through chemical synthesis is based on petroleum derivatives, resulting in concerns for the environmental complication and energy crisis, and increasing attention has been attracted to the eco-friendly and renewable bio-based synthesis for carboxylate production. The efficient and specific export of target carboxylic acids through the microbial membrane is essential for high productivity, yield, and titer of bio-based carboxylates. Therefore, understanding the characteristics, regulations, and efflux mechanisms of carboxylate transporters will efficiently increase industrial biotechnological production of carboxylic acids. Several transporters from fungi have been reported and used for improved synthesis of target products. The transport activity and substrate specificity are two key issues that need further improvement in the application of carboxylate transporters. This review presents developments in the structural and functional diversity of carboxylate transporters, focusing on the modification and regulation of carboxylate transporters to alter the transport activity and substrate specificity, providing new strategy for transporter engineering in constructing microbial cell factory for carboxylate production. KEY POINTS: • Structures of multiple carboxylate transporters have been predicted. • Carboxylate transporters can efficiently improve production. • Modification engineering of carboxylate transporters will be more popular in the future.

Consolidated bioprocessing for bioethanol production by metabolically engineered cellulolytic fungus Myceliophthora thermophila.

Using cellulosic ethanol as fuel is one way to help achieve the world's decarbonization goals. However, the economics of the present technology are unfavorable, especially the cost of cellulose degradation. Here, we reprogram the thermophilic cellulosic fungus Myceliophthora thermophila to directly ferment cellulose into ethanol by mimicking the aerobic ethanol fermentation of yeast (the Crabtree effect), including optimizing the synthetic pathway, enhancing the glycolytic rate, inhibiting mitochondrial NADH shuttles, and knocking out ethanol consumption pathway. The final engineered strain produced 52.8 g/L ethanol directly from cellulose, and 39.8 g/L from corncob, without the need for any added cellulase, while the starting strain produced almost no ethanol. We also demonstrate that as the ethanol fermentation by engineered M. thermophila increases, the composition and expression of cellulases that facilitate the degradation of cellulose, especially cellobiohydrolases, changes. The simplified production process and significantly increased ethanol yield indicate that the fungal consolidated bioprocessing technology that we develop here (one-step, one-strain ethanol production) is promising for fueling sustainable carbon-neutral biomanufacturing in the future.

Dissecting key residues of a C4-dicarboxylic acid transporter to accelerate malate export in Myceliophthora.

Efficient transporters are necessary for high concentration and purity of desired products during industrial production. In this study, we explored the mechanism of substrate transport and preference of the C4-dicarboxylic acid transporter AoMAE in the fungus Myceliophthora thermophila, and investigated the roles of 18 critical amino acid residues within this process. Among them, the residue Arg78, forming a hydrogen bond network with Arg23, Phe25, Thr74, Leu81, His82, and Glu94 to stabilize the protein conformation, is irreplaceable for the export function of AoMAE. Furthermore, varying the residue at position 100 resulted in changes to the size and shape of the substrate binding pocket, leading to alterations in transport efficiencies of both malic acid and succinic acid. We found that the mutation T100S increased malate production by 68%. Using these insights, we successfully generated an AoMAE variant with mutation T100S and deubiquitination, exhibiting an 81% increase in the selective export activity of malic acid. Simply introducing this version of AoMAE into M. thermophila wild-type strain increased production of malic acid from 1.22 to 54.88 g/L. These findings increase our understanding of the structure-function relationships of organic acid transporters and may accelerate the process of engineering dicarboxylic acid transporters with high efficiency. KEY POINTS: • This is the first systematical analysis of key residues of a malate transporter in fungi. • Protein engineering of AoMAE led to 81% increase of malate export activity. • Arg78 was essential for the normal function of AoMAE in M. thermophila. • Substitution of Thr100 affected export efficiency and substrate selectivity of AoMAE.

Coordination of consolidated bioprocessing technology and carbon dioxide fixation to produce malic acid directly from plant biomass in Myceliophthora thermophila.

BACKGROUND: Consolidated bioprocessing (CBP) technique is a promising strategy for biorefinery construction, producing bulk chemicals directly from plant biomass without extra hydrolysis steps. Fixing and channeling CO2 into carbon metabolism for increased carbon efficiency in producing value-added compounds is another strategy for cost-effective bio-manufacturing. It has not been reported whether these two strategies can be combined in one microbial platform. RESULTS: In this study, using the cellulolytic thermophilic fungus Myceliophthora thermophila, we designed and constructed a novel biorefinery system DMCC (Direct microbial conversion of biomass with CO2 fixation) through incorporating two CO2 fixation modules, PYC module and Calvin-Benson-Bassham (CBB) pathway. Harboring the both modules, the average rate of fixing and channeling 13CO2 into malic acid in strain CP51 achieved 44.4, 90.7, and 80.7 mg/L/h, on xylose, glucose, and cellulose, respectively. The corresponding titers of malic acid were up to 42.1, 70.4, and 70.1 g/L, respectively, representing the increases of 40%, 10%, and 7%, respectively, compared to the parental strain possessing only PYC module. The DMCC system was further improved by enhancing the pentose uptake ability. Using raw plant biomass as the feedstock, yield of malic acid produced by the DMCC system was up to 0.53 g/g, with 13C content of 0.44 mol/mol malic acid, suggesting DMCC system can produce 1 t of malic acid from 1.89 t of biomass and fix 0.14 t CO2 accordingly. CONCLUSIONS: This study designed and constructed a novel biorefinery system named DMCC, which can convert raw plant biomass and CO2 into organic acid efficiently, presenting a promising strategy for cost-effective production of value-added compounds in biorefinery. The DMCC system is one of great options for realization of carbon neutral economy.

Transcriptional Profiling of Myceliophthora thermophila on Galactose and Metabolic Engineering for Improved Galactose Utilization.

Efficient biological conversion of all sugars from lignocellulosic biomass is necessary for the cost-effective production of biofuels and commodity chemicals. Galactose is one of the most abundant sugar in many hemicelluloses, and it will be important to capture this carbon for an efficient bioconversion process of plant biomass. Thermophilic fungus Myceliophthora thermophila has been used as a cell factory to produce biochemicals directly from renewable polysaccharides. In this study, we draw out the two native galactose utilization pathways, including the Leloir pathway and oxido-reductive pathway, and identify the significance and contribution of them, through transcriptional profiling analysis of M. thermophila and its mutants on galactose. We find that galactokinase was necessary for galactose transporter expression, and disruption of galK resulted in decreased galactose utilization. Through metabolic engineering, both galactokinase deletion and galactose transporter overexpression can activate internal the oxido-reductive pathway and improve the consumption rate of galactose. Finally, the heterologous galactose-degradation pathway, De Ley-Doudoroff (DLD) pathway, was successfully integrated into M. thermophila, and the consumption rate of galactose in the engineered strain was increased by 57%. Our study focuses on metabolic engineering for accelerating galactose utilization in a thermophilic fungus that will be beneficial for the rational design of fungal strains to produce biofuels and biochemicals from a variety of feedstocks with abundant galactose.

A putative transcription factor LFC1 negatively regulates development and yield of winter mushroom.

Basidioma is the fruiting body of mushroom species. The deep understanding on the mechanism of basidioma development is valuable for mushroom breeding and cultivation. From winter mushroom (Flammulina velutipes), one of the top five industrially cultivated mushrooms, a novel putative Zn(II)2Cys6 transcription factor LFC1 with negative regulatory function in basidioma development was identified. The transcript level of lfc1 was dramatically decreased during basidioma development. Neither overexpression nor knockdown of lfc1 affected hyphal vegetative growth. However, knockdown of lfc1 could promote basidioma development and shorten cultivation time by 2 days, while overexpression of lfc1 delayed the optimal harvest time by 3 days. In the lfc1 knockdown strain, in which the lfc1 expression was reduced by 72%, mushroom yield and biological efficiency could be increased at least by 24%. Knockdown of lfc1 did not affect the shape of caps but significantly increased basidioma length and number, while its overexpression did not affect basidioma length but dramatically reduced basidioma number. In addition, rather than producing basidiomata with round caps as in wild type, the caps of basidiomata in the lfc1 overexpression mutants were significantly larger and the cap edge was wrinkled. RNA-seq analysis revealed that 455 genes had opposite transcriptional responses to lfc1 overexpression and knockdown. Some of them were previously reported as genes involved in basidioma development, including 3 hydrophobin encoding genes, 2 lectin encoding genes, FVFD16, an Eln2 ortholog encoding gene, and 3 genes encoding membrane components. As LFC1 homologs are widely present in mushroom species, lfc1 can be useful in mushroom breeding.Key Points• A novel transcription factor LFC1 negatively regulates fruiting in winter mushroom• LFC1 regulated transcription of more than 400 genes.• Reduction of LFC1 expression could shorten cultivation time and increase yield.• lfc1 could be a potentially useful reference gene for mushroom breeding.

A WD40 Protein Encoding Gene Fvcpc2 Positively Regulates Mushroom Development and Yield in Flammulina velutipes.

Ascomycota and Basidiomycota are two closely related phyla and fungi in two phyla share some common morphological developmental process during fruiting body formation. In Neurospora crassa, the Gß-like protein CPC-2 with a seven-WD40 repeat domain was previously reported. By transforming CPC-2 ortholog encoding genes, from 7 different fungal species across Ascomycota and Basidiomycota, into the cpc-2 deletion mutant of N. crassa, we demonstrate that all tested CPC-2 ortholog genes were able to complement the defects of the cpc-2 deletion mutant in sexual development, indicating that CPC-2 proteins from Ascomycota and Basidiomycota have the similar cellular function. Using Flammulina velutipes as a model system for mushroom species, the CPC-2 ortholog FvCPC2 was characterized. Fvcpc2 increased transcription during fruiting body development. Knockdown of Fvcpc2 by RNAi completely impaired fruiting body formation. In three Fvcpc2 knockdown mutants, transcriptional levels of genes encoding adenylate cyclase and protein kinase A catalytic subunit were significantly lower and colony growth became slower than wild type. The addition of cAMP or the PKA-activator 8-Bromo-cAMP into the medium restored the Fvcpc2 knockdown mutants to the wild-type colony growth phenotype, suggesting that the involvement of cAMP production in the regulatory mechanisms of FvCPC2. Knockdown of Fvcpc2 also weakened transcriptional responses to sexual development induction by some genes related to fruiting body development, including 4 jacalin-related lectin encoding genes, 4 hydrophobin encoding genes, and 3 functionally-unknown genes, suggesting the participation of these genes in the mechanisms by which FvCPC2 regulates fruiting body development. All three Fvcpc2 overexpression strains displayed increased mushroom yield and shortened cultivation time compared to wild type, suggesting that Fvcpc2 can be a promising reference gene for Winter Mushroom breeding. Since the orthologs of FvCPC2 were highly conserved and specifically expressed during fruiting body development in different edible mushrooms, genes encoding FvCPC2 orthologs in other mushroom species also have potential application in breeding.

A Single Transcription Factor (PDD1) Determines Development and Yield of Winter Mushroom (Flammulina velutipes).

Most of the edible mushrooms cannot be cultivated or have low yield under industrial conditions, partially due to the lack of knowledge on how basidioma (fruiting body) development is regulated. From winter mushroom (Flammulina velutipes), one of the most popular industrially cultivated mushrooms, a transcription factor, PDD1, with a high-mobility group (HMG)-box domain was identified based on its increased transcription during basidioma development. pdd1 knockdown by RNA interference affected vegetative growth and dramatically impaired basidioma development. A strain with an 89.9% reduction in the level of pdd1 transcription failed to produce primordia, while overexpression of pdd1 promoted basidioma development. When the transcriptional level of pdd1 was increased to 5 times the base level, the mushroom cultivation time was shortened by 9.8% and the yield was increased by at least 33%. RNA sequencing (RNA-seq) analysis revealed that pdd1 knockdown downregulated 331 genes and upregulated 463 genes. PDD1 positively regulated several genes related to fruiting, including 6 pheromone receptor-encoding genes, 3 jacalin-related lectin-encoding genes, FVFD16, and 2 FVFD16 homolog-encoding genes. PDD1 is a novel transcription factor with regulatory function in basidioma development found in industrially cultivated mushrooms. Since its orthologs are widely present in fungal species of the Basidiomycota phylum, PDD1 might have important application prospects in mushroom breeding.IMPORTANCE Mushrooms are sources of food and medicine and provide abundant nutrients and bioactive compounds. However, most of the edible mushrooms cannot be cultivated commercially due to the limited understanding of basidioma development. From winter mushroom (Flammulina velutipes; also known as Enokitake), one of the most commonly cultivated mushrooms, we identified a novel transcription factor, PDD1, positively regulating basidioma development. PDD1 increases expression during basidioma development. Artificially increasing its expression promoted basidioma formation and dramatically increased mushroom yield, while reducing its expression dramatically impaired its development. In its PDD1 overexpression mutants, mushroom number, height, yield, and biological efficiency were significantly increased. PDD1 regulates the expression of some genes that are important in or related to basidioma development. PDD1 is the first identified transcription factor with defined functions in mushroom development among commercially cultivated mushroom species, and it might be useful in mushroom breeding.

Small GTPases and Stress Responses of vvran1 in the Straw Mushroom Volvariella volvacea.

Small GTPases play important roles in the growth, development and environmental responses of eukaryotes. Based on the genomic sequence of the straw mushroom Volvariella volvacea, 44 small GTPases were identified. A clustering analysis using human small GTPases as the references revealed that V. volvacea small GTPases can be grouped into five families: nine are in the Ras family, 10 are in the Rho family, 15 are in the Rab family, one is in the Ran family and nine are in the Arf family. The transcription of vvran1 was up-regulated upon hydrogen peroxide (H2O2) stress, and could be repressed by diphenyleneiodonium chloride (DPI), a NADPH oxidase-specific inhibitor. The number of vvran1 transcripts also increased upon cold stress. Diphenyleneiodonium chloride, but not the superoxide dismutase (SOD) inhibitor diethy dithiocarbamate (DDC), could suppress the up-regulation of vvran1 gene expression to cold stress. These results combined with the high correlations between gene expression and superoxide anion (O2(-)) generation indicated that vvran1 could be one of the candidate genes in the downstream of O2(-) mediated pathways that are generated by NADPH oxidase under low temperature and oxidative stresses.

The Accordant Trend of Both Parameters (rgs Expression and cAMP Content) Follows the Pattern of Development of Fruiting Body in Volvariella volvacea.

The formation of fruiting body in Volvariella volvacea is affected by endogenous genes and environmental factors. However, its regulation at a molecular level is still poorly understood. To study the genes involved in the formation of fruiting body, we cloned a new regulator of the G protein signaling (RGS) encoding gene (rgs) from V. volvacea. Phylogenetic analysis showed that RGS in V. volvacea and other basidiomycete RGS proteins from Schizophyllum commune and Coprinus cinereus belong to the same clade. In addition, we assayed intracellular cAMP content in the three developmental stages (mycelium, fruiting body primordia, and button). We also found that the expression of rgs was highly positively correlated to the content of intracellular cAMP during fruiting body formation. The conserved protein sequences and expression of rgs, together with high concent of cAMP at primordia tissue, suggested that rgs gene and cAMP may play a crucial role in fruiting body formation in V. volvacea.

Structural Variation (SV) Markers in the Basidiomycete Volvariella volvacea and Their Application in the Construction of a Genetic Map.

Molecular markers and genetic maps are useful tools in genetic studies. Novel molecular markers and their applications have been developed in recent years. With the recent advancements in sequencing technology, the genomic sequences of an increasingly great number of fungi have become available. A novel type of molecular marker was developed to construct the first reported linkage map of the edible and economically important basidiomycete Volvariella volvacea by using 104 structural variation (SV) markers that are based on the genomic sequences. Because of the special and simple life cycle in basidiomycete, SV markers can be effectively developed by genomic comparison and tested in single spore isolates (SSIs). This stable, convenient and rapidly developed marker may assist in the construction of genetic maps and facilitate genomic research for other species of fungi.

Gene expression profiling reveals large regulatory switches between succeeding stipe stages in Volvariella volvacea.

The edible mushroom Volvariella volvacea is an important crop in Southeast Asia and is predominantly harvested in the egg stage. One of the main factors that negatively affect its yield and value is the rapid transition from the egg to the elongation stage, which has a decreased commodity value and shelf life. To improve our understanding of the changes during stipe development and the transition from egg to elongation stage in particular, we analyzed gene transcription in stipe tissue of V. volvacea using 3'-tag based digital expression profiling. Stipe development turned out to be fairly complex with high numbers of expressed genes, and regulation of stage differences is mediated mainly by changes in expression levels of genes, rather than on/off modulation. Most explicit is the strong up-regulation of cell division from button to egg, and the very strong down-regulation hereof from egg to elongation, that continues in the maturation stage. Button and egg share cell division as means of growth, followed by a major developmental shift towards rapid stipe elongation based on cell extension as demonstrated by inactivation of cell division throughout elongation and maturation. Examination of regulatory genes up-regulated from egg to elongation identified three potential high upstream regulators for this switch. The new insights in stipe dynamics, together with a series of new target genes, will provide a sound base for further studies on the developmental mechanisms of mushroom stipes and the switch from egg to elongation in V. volvacea in particular.