Backgroundprp operon usually comprises prpB, prpC, prpD and prpE genes, encoding 2-methylisocitrate (2-MIC) lyase, 2-methylcitrate (2-MC) synthase, 2-MC dehydratase and propionyl-CoA synthetase, respectively, which constitute the pathway 2-methylcitrate cycle (2-MCC) well-known for microbial propionate metabolism. Acetic acid bacteria (AAB) represent a group of obligate aerobic and Gram-negative microorganisms. Attributed to the robust ethanol-oxidating and acetic acid-resisting abilities, vinegar production stands for an important AAB's industrial application. Previously, we unveiled the first AAB's prp operon in Acetobacter pasteurianus CGMCC 1.41-a vinegar-brewing strain, and its up-regulation during acetic acid fermentation, suggesting the genes' potential contribution to the adaptive processes of this strain. In this study, the prevalence of prp genes among AAB were analyzed, followed by constructing six markerless deletion mutants to investigate the genes' role in carbon source metabolism and acetic acid resistance in A. pasteurianus CGMCC 1.41.ResultsThe prp gene cluster lacking prpE was found widespread in the genus Acetobacter. A gene encoding PrpE-homologous acetyl-CoA synthetase, designated prpE', was discovered to participate the formation of the putative operon in many AAB genomes. Growth experiments revealed the ability to utilize propionate as a carbon source of A. pasteurianus CGMCC 1.41, which simultaneously required the function of PrpB, PrpC and PrpD. A role of PrpD or PrpB in promoting ethanol assimilation was observed, whereas PrpE' appeared to be important for metabolizing both ethanol and acetic acid as carbon sources. PrpB, PrpC and PrpD also exerted a positive effect on glycerol metabolism. Moreover, the integrity of the 2-MCC pathway exhibited a great importance upon the initiation of the bioreaction catalyzed by PrpC. Finally, PrpE' was assumed to be critical for an acetic acid-resisting process-acetic acid overoxidation, which might be potentially enhanced by PrpD as well.ConclusionThe present work expands the knowledge to the prp operon in the context of AAB, which also indicates the significance of gaining deeper insight into the specific roles of relevant genes in this group of bacteria.

Benzenediol lactones (BDL) and Monascus azaphilone pigments (MonAzP) are different classes of bioactive polyketides produced by distinct pathways. BDL are produced by highly reducing polyketide synthase (hrPKS) - nonreducing PKS (nrPKS) pairs where the starter acyl transferase (SAT) domain of the nrPKS recruits an advanced starter unit provided by the hrPKS. In contrast, the MonAzP nrPKS does not use a hrPKS partner: instead, its SAT domain selects acetyl-CoA as the priming unit. Here, we reconfigured the MonAzP nrPKS to collaborate in a dual-PKS system by replacing its SAT domain with that of a BDL nrPKS. This shifted the product spectrum from benzaldehydes to novel α-pyrones in the chimeric system. Our work highlights that SAT domain engineering may enforce noncognate hrPKS-nrPKS collaborations even for solitary nrPKSs. Nevertheless, the derailment of the native cyclization and product release routines indicates that domain compatibility must be considered when engineering to produce unnatural polyketides.

Abstract Monascus spp. are economically important filamentous fungi that have been utilized in the production of beneficial metabolites such as Monascus pigments and monacolin K, as well as in the brewing of some Asian fermented foods. The delimitation of Monascus species has traditionally relied on phenotypic traits; however, this morphological classification approach is susceptible to subjective judgments and variations in cultural conditions and also may not necessarily be related to the actual genetic relationship. Consequently, synonymy and misidentification frequently occur in Monascus taxonomy, highlighting the urgent need for a convenient and reliable classification system for this genus. In this study, a phylogenetic analysis of 82 representative Monascus strains, encompassing all previously recognized species of the genus, was conducted based on the concordance of five gene genealogies ( BenA , CaM , ITS , LSU , and RPB2 ) to clarify species delimitation and resolve phylogenetic relationships within Monascus . The results revealed that the genus Monascus is resolved into 11 species, which are clustered into two sections: Floridani (including M. argentinensis , M. flavipigmentosus , M. floridanus , M. lunisporas , M. mellicola , M. pallens , and M. recifensis ) and Rubri (including M. pilosus , M. purpureus , M. ruber , and M. sanguineus ). M. pilosus and M. sanguineus were reaffirmed as distinct species due to their well-supported and divergent phylogenetic lineages. Additionally, M. albidulus , M . anka , M. barkeri , and M. fumeus are synonymized with M. pilosus , while M. aurantiacus and M. rutilus are synonyms of M. purpureus. Finally, a comprehensive list of accepted Monascus species along with their corresponding barcode sequence data is provided.

Wuyi Hongqu (WYH), also called black-skin-red-koji, which has been utilizing as a fermentation starter for more than one thousand years in China, is a symbiotic combination of Monascus spp. and Aspergillus niger formed through long-term application and domestication. In this study, the strains of Monascus purpureus and A. niger isolated from WYH samples were used to investigate their mutual influence, especially the effects on three main secondary metabolites from M. purpureus, Monascus pigments (MPs), monacolin K (MK), and citrinin (CIT), using a double-sided Petri dish (DSPD). The results showed that co-cultivation of M. purpureus and A. niger strains was favorable to increase the MPs production while inhibiting the CIT production by M. purpureus, especially when M. purpureus strains (M1-1 or M9) were co-cultivated with certain A. niger strains (An1-2 or An9), respectively, and both Monascus strains hardly produced detectable CIT. The expression levels of CIT-related genes in M. purpureus M1-1 or M9 were greatly restricted when co-cultivated with A. niger An1-2 or An9 confirmed by RT-qPCR. This study provides important insights into the selection of WYH production strains and the effects of fungal interactions.

Phenyllactic acid (PLA) is a broad-spectrum antimicrobial substance with broad application prospects in the food industry. In our previous research, we isolated and screened a strain of acetic acid bacteria (AAB) from vinegar mash, Gluconacetobacter tumulisoli FBFS 97, which is the first reported AAB strain with the PLA production capacity. In current study, in order to increase the PLA production of the FBFS 97 strain, a strong promoter, Pkan (promoter of kanamycin resistance gene), suitable for the FBFS 97 strain, was screened using enhanced green fluorescent protein (EGFP) as a reporter. Then five related genes with PLA biosynthesis in the FBFS 97 strain, including the chorismate synthase gene aroc, the prephenate dehydratase gene pheA, the glucose dehydrogenase gene gdh, the formate dehydrogenase gene fdh and the glycerate dehydrogenase gene gldh were overexpressed using the Pkanpromoter, respectively. The results revealed that except the gldh overexpression strain, PLA production of other mutants were improved to 125.9 %, 140.2 %, 130 % and 113 % comparing to that (56.85 mg/L) of the FBFS 97 strain, respectively. This study not only lays the foundation for constructing the high-producing PLA engineering strain using the FBFS 97, but also provides a novel insight for better understanding the PLA biosynthesis pathway of microorganisms.

Monacolin K (MK), a secondary metabolite produced by Monascus spp. with the ability to inhibit cholesterol production, is structurally identical to lovastatin produced by Aspergillus terreus. In the lovastatin biosynthetic pathway, the polyketide synthase (PKS) encoded by lovB must work together with the enoyl reductase encoded by lovC to ensure lovastatin production. However, it is unclear whether mokA and mokE in the MK gene cluster of Monascus spp., both of which are highly homologous to lovB and lovC, respectively, also have the same functions for MK biosynthesis. In the current study, the high-yielding MK M. pilosus MS-1 was used as the research object, and it was found that the enoyl reductase domain of MokA may be non-functional due to the lack of amino acids at active sites, a function that may be compensated for by MokE in the MK synthesis pathway. Then, the mokE-deleted (ΔmokE), -complemented (ΔmokE::mokE), and -overexpressed (PgpdA-mokE) strains were constructed, and the results showed that ΔmokE did not produce MK, and ΔmokE::mokE restored MK synthesis, while the ability of PgpdA-mokE to produce MK was increased by 32.1% compared with the original strain MS-1. These results suggest that the MokA synthesized by Monascus spp. must be assisted by MokE to produce MK, just as lovastatin produced by A. terreus, which provides clues for further genetic engineering to improve the yield of MK in Monascus spp.

Sensory analysis is a very powerful and useful tool that is used for a variety of foods. But for vinegar, the relevant sensory evaluation system is not satisfactory since there are still some issues, such as the tendency for score conservatism, descriptor redundancy, sensory fatigue and other cognitive issues. In this review, the theory of the sensory evaluation of vinegar is first introduced, and then the application of sensory evaluation is summarized for vinegar, especially for Italian Traditional Balsamic Vinegar. By improving the scoring system and enhancing the tasting conditions, the reliability of vinegar sensory evaluation can be further increased and provide a solid support for vinegar quality control and market promotion.

Acetic acid bacteria (AAB) are a group of bacteria, most of which can produce pigments. However, the mechanism of pigment production by AAB is unclear. A strain of AAB, Gluconacetobacter tumulisoli FBFS 97, which can produce a large amount of brown pigment (BP), was isolated in our previous research. In the current study, it was found that the BP yield of the FBFS 97 strain was enhanced in the presence of tyrosine, and an intermediate of melanin, L-3,4-dihydroxyphenylalanine (L-DOPA), was identified using ultra-performance liquid chromatography–quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). The structural properties of BP were analyzed by pyrolysis gas chromatography–mass spectrometry (Py-GC-MS). All these analyses suggest that BP may be eumelanin, a type of melanin. Then, the eumelanin biosynthetic pathway was investigated in the FBFS 97 strain, and three related genes with eumelanin including pheA, yfiH, and phhB in its genome were found and knocked out, respectively. The results showed that eumelanin production increased 1.3-fold in the pheA deletion mutant compared to the wild-type FBFS 97 strain, but when either yfiH or phhB was knocked out, the eumelanin production in the mutants was the same as that in the wild-type FBFS 97 strain. Finally, a possible biosynthetic pathway for eumelanin in the FBFS 97 strain is proposed.

Monascus, a genus of fungi known for its fermentation capability and production of bioactive compounds, such as Monascus azaphilone pigments and Monacolin K, have received considerable attention because of their potential in biotechnological applications. Understanding the genetic basis of these metabolic pathways is crucial for optimizing the fermentation and enhancing the yield and quality of these products. However, Monascus spp. are not model fungi, and knowledge of their genetics is limited, which is a great challenge in understanding physiological and biochemical phenomena at the genetic level. Since the first application of particle bombardment to explore gene function, it has become feasible to link the phenotypic variation and genomic information on Monascus strains. In recent decades, accurate gene editing assisted by genomic information has provided a solution to analyze the functions of genes involved in the metabolism and development of Monascus spp. at the molecular level. This review summarizes most of the genetic manipulation tools used in Monascus spp. and emphasizes Agrobacterium tumefaciens-mediated transformation and nuclease-guided gene editing, providing comprehensive references for scholars to select suitable genetic manipulation tools to investigate the functions of genes of interest in Monascus spp.

wetA, one of the conidiation center regulatory genes in many filamentous fungi, plays an important role in promoting asexual spores (conidia) maturation. Our recent research has found that knocking out or overexpressing MrwetA (a homolog of wetA) in Monascus ruber M7 does not affect the development of its asexual spores like other fungi, but both repress the development of its sexual spores (ascospores). However, the mechanism remains unclear. In this study, the function of MrwetA on sexual reproduction and secondary metabolism in M. ruber M7 was confirmed by a complementary experiment. Moreover, the regulatory roles of MrwetA in modulating the expression of genes involved in sexual reproduction, meiosis, and biosynthesis of Monascus pigment and citrinin were analyzed based on the transcriptional data. These results not only contribute to clarifying the regulation of the reproduction and secondary metabolism of Monascus spp., but also to enriching the regulation molecular mechanism of reproduction in filamentous fungi.

Blue light, as an important environmental factor, greatly affects the production of Monascus pigments (MPs) and citrinin in Monascus spp.. In this study, the deletion, complementation, and overexpression mutants of MrcreD from Monascus ruber M7, which encodes an arrestin-like protein, were constructed and cultivated on PDA (Potato dextrose agar) medium to study the effects of blue light on MPs and citrinin production. The results revealed that blue light inhibited the formation of cleistothecia, conidia, and the production of MPs and citrinin in M. ruber M7. However, under blue light, in contrast to M. ruber M7, MrcreD-overexpressing strain displayed increased production of extracellular yellow pigments and intracellular orange pigments, whereas MrcreD-deleted strain showed enhanced production of intracellular yellow and orange pigments. Then, the extracellular citrinin production decreased in both mutants. The RT-qPCR results demonstrated that compared to M. ruber M7, overexpressing MrcreD increased the expression of genes involved in MPs biosynthesis, and decreased the genes involved in citrinin biosynthesis, while deleting MrcreD increased the expression of citrinin-relative genes. This is the first time that the functions of MrcreD gene in filamentous fungi have been researched under blue light, and it provides a strategy for exploring complex light-regulatory systems in filamentous fungi.

Diphenyl ethers (DPEs) are produced by filamentous fungi using polyketide synthases (PKSs) directly, or via Cu oxidase-catalyzed oxidative rearrangements of benzophenone intermediates. Here, we use heterologous expression to reveal a third route towards DPEs in Preussia isomera that relies on an oxidative multienzyme cascade to convert a PKS-generated, ester-linked didepside to depsidones and further to DPEs, and apply comparative genomics to identify conserved biosynthetic gene clusters for this pathway in multiple fungi. The distribution of DPE products is modulated by the expression chassis upon pathway reconstitution. Among the post-PKS enzymes, the DpeH tyrosinase shows considerable substrate promiscuity towards synthetic DPE analogues. By creating hybrid enzymes with a DpeH orthologue from Aspergillus nidulans, we identify the C-terminal region of DpeH to alter substrate recognition. Our work highlights an evolutionarily conserved way to produce DPEs, and provides enzymatic tools to generate DPE analogues with broad spectrum antibiotic activity against multidrug-resistant human pathogens.

Monascus pilosus has been used to produce lipid-lowering drugs rich in monacolin K (MK) for a long period. Genome mining reveals there are still many potential genes worth to be explored in this fungus. Thereby, efficient genetic manipulation tools will greatly accelerate this progress. In this study, we firstly developed the protocol to prepare protoplasts for recipient of CRISPR/Cas9 system. Subsequently, the vector and donor DNA were co-transformed into recipients (10 6 protoplasts/mL) to produce 60–80 transformants for one test. Three genes ( mpclr4 , mpdot1 , and mplig4 ) related to DNA damage response (DDR) were selected to compare the gene replacement frequencies (GRFs) of Agrobacterium tumefaciens -mediated transformation (ATMT) and CRISPR/Cas9 gene editing system (CGES) in M. pilosus MS-1. The results revealed that GRF of CGES was approximately five times greater than that of ATMT, suggesting that CGES was superior to ATMT as a targeting gene editing tool in M. pilosus MS-1. The inactivation of mpclr4 promoted DDR via the non-homologous end-joining (NHEJ) and increased the tolerances to DNA damaging agents. The inactivation of mpdot1 blocked DDR and led to the reduced tolerances to DNA damaging agents. The inactivation of mplig4 mainly blocked the NHEJ pathway and led to obviously reduced tolerances to DNA damaging agents. The submerged fermentation showed that the ability to produce MK in strain Δ mpclr4 was improved by 52.6% compared to the wild type. This study provides an idea for more effective exploration of gene functions in Monascus strains. Key points • A protocol of high-quality protoplasts for CGES has been developed in M. pilosus. • The GRF of CGES was about five times that of ATMT in M. pilosus. • The yield of MK for Δmpclr4 was enhanced by 52.6% compared with the wild type.

Increasing data suggested that histone methylation modification plays an important role in regulating biosynthesis of secondary metabolites (SMs). Monascus spp. have been applied to produce hypolipidemic drug lovastatin (also called monacolin K, MK) and edible Monascus-type azaphilone pigments (MonAzPs). However, little is known about how histone methylation regulates MK and MonAzPs. In this study, we constructed H3K9 methyltransferase deletion strain ΔMpDot1 and H4K20 methyltransferase deletion strain ΔMpSet9 using Monascus pilosus MS-1 as the parent. The result showed that deletion of MpDot1 reduced the production of MK and MonAzPs, and deletion of MpSet9 increased MonAzPs production. Real-time quantitative PCR (RT-qPCR) showed inactivation of mpdot1 and mpset9 disturbed the expression of genes responsible for the biosynthesis of MK and MonAzPs. Western blot suggested that deletion of MpDot1 reduced H3K79me and H4K16ac, and deletion of MpSet9 decreased H4K20me3 and increased H4pan acetylation. Chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) showed ΔMpDot1 strain and ΔMpSet9 strain reduced the enrichment of H3K79me2 and H4K20me3 in the promoter regions of key genes for MK and MonAzPs biosynthesis, respectively. These results suggested that MpDot1 and MpSet9 affected the synthesis of SMs by regulating gene transcription and histone crosstalk, providing alternative approach for regulation of lovastatin and MonAzPs.

Polyketides with the isochroman-3-one pharmacophore are rare among fungal natural products as their biosynthesis requires an unorthodox S-type aromatic ring cyclization. Genome mining uncovered a conserved gene cluster in select leotiomycetous fungi that encodes the biosynthesis of cytosporones, including isochroman-3-one congeners. Combinatorial biosynthesis in total biosynthetic and biocatalytic formats in Saccharomyces cerevisiae and in vitro reconstitution of key reactions with purified enzymes revealed how cytosporone structural and bioactivity diversity is generated. The S-type acyl dihydroxyphenylacetic acid (ADA) core of cytosporones is assembled by a collaborating polyketide synthase pair. Thioesterase domain-catalyzed transesterification releases ADA esters, some of which are known Nur77 modulators. Alternatively, hydrolytic release allows C6 hydroxylation by a flavin-dependent monooxygenase, yielding a trihydroxybenzene moiety. Reduction of the C9 carbonyl by a short chain dehydrogenase/reductase initiates isochroman-3-one formation, affording cytosporones with cytotoxic and antimicrobial activity. Enoyl di- or trihydroxyphenylacetic acids are generated as shunt products, while isocroman-3,4-diones are formed by autoxidation. The cytosporone pathway offers novel polyketide biosynthetic enzymes for combinatorial synthetic biology to advance the production of "unnatural" natural products for drug discovery.

Vam7 (vacuolar morphogenesis 7), a Qc-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) that mediates vacuolar fusion, is involved in vacuolar morphogenesis, vegetative growth, sexual and asexual reproduction, and compartmentalized biosynthesis of secondary metabolites in various filamentous fungi. In this study, the homolog of vam7 gene, mrvam7, in Monascus ruber M7 was functionally characterized through gene modification and transcriptome analysis. The results showed that the mrvam7 complementation and overexpression strains exhibited phenotypic similarities to M. ruber M7, whereas the mrvam7 null strain exhibited fragmented vacuoles, slowed growth and diminished sexual and asexual sporulation. Furthermore, the mrvam7 knockout strain displayed a notable increase in citrinin production alongside a significant decrease in Monascus pigments production. Transcriptome data revealed that the deletion of mrvam7 altered the expression levels of several genes involved in sexual and asexual reproduction, carbon and nitrogen source utilization, and secondary metabolites biosynthetic gene clusters. Collectively, these findings underscore the critical role of mrvam7-mediated vacuolar fusion in growth, development, and the compartmentalized biosynthesis and distribution of secondary metabolites in M ruber M7. This study provides novel insights into the mechanisms underlying of compartmentalized secondary metabolites biosynthesis in Monascus species.

Monascus azaphilone pigments (MonAzPs) are beneficial secondary metabolites secreted by Monascus spp., which have great potentials to be used in industrial fields besides food industry. But there is very little knowledge about their regulatory mechanism, which limits the large-scale production and utilization of MonAzPs. In this study, we firstly studied the effect on MonAzPs production by inhibiting and enhancing histone deacetylase gene hos2 (Mrhos2) expression in Monascus ruber. UPLC detection revealed that inactivation of Mrhos2 dramatically decreased MonAzPs production, while overexpression of Mrhos2 significantly increased MonAzPs production. Transcriptional level analysis shows Mrhos2 regulates expression of MonAzPs biosynthesis genes. Immunoaffinity enrichment assisted mass spectrometry assay reveals deletion of Mrhos2 perturbs lysine acetylation of enzymes in the metabolic pathway of acetyl-CoA and malonyl-CoA. Collectively, transcription activation and protein acetylation modification mediated by MrHos2 are involved in regulating the MonAzPs production, and MrHos2 can be used as a target to increase MonAzPs production.

Tender coconut (TC) is popular around the world. Postharvest storage of TC leads to a decline in its appearance quality and flavor in both liquid endosperm (LE) and solid endosperm (SE). While LE is the most consumed part and remains in a liquid state, SE is the only cellular tissue directly connected to LE and may be the main contributor to flavor deterioration during storage. This study focused on investigating SE changes during TC storage at 25 degrees C using computed tomographic technology, transcriptome and metabolome analyses. The results showed increased thickness and density, elevated protein and fat contents, and decreased reducing and soluble sugars in SE of TC during storage. Integrated transcriptome and metabolome analysis revealed that these changes were mainly associated with the gene transcription levels involved in amino acid, carbohydrate and lipid metabolisms, along with specific metabolites. These findings offer valuable insights for controlling TC quality during storage.

Monascus pigments (MPs), a class of secondary metabolites produced by Monascus spp., can be classified into yellow, orange, and red MPs according to their differences in the wavelength of the maximum absorption. However, the biosynthetic sequence and cellular biosynthesis mechanism of different MPs components are still not yet completely clear in Monascus spp. In this study, the subcellular localization of five MPs synthases was investigated using fluorescent protein fusion expression. The results revealed that the proteins encoded by the MPs biosynthetic gene cluster were compartmentalized in various subcellular locations, including the mitochondrial polyketide synthase MrPigA, cytosolic enzymes consisting of the ketoreductase MrPigC, the oxidoreductase MrPigE, and the monooxygenase MrPigN, and the cell-wall-bound oxidoreductase MrPigF. Moreover, the correct localization of MrPigF to the cell wall was crucial for the synthesis of orange MPs. Lastly, we discussed the compartmentalized biosynthetic pathway of MPs. This study will not only be helpful in clarifying the biosynthetic sequence and biosynthesis mechanism of different MPs but also provides new insights into the cellular biosynthesis of secondary metabolites in filamentous fungi.