CRISPR/Cas9 system is a suitable gene targeting editing tool to filamentous fungus Monascus pilosus.

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 (106 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 Δmpclr4 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.

Mrhst4 gene, coding for NAD+-dependent deacetylase is involved in citrinin production of Monascus ruber.

AIMS: In this study, Mrhst4, encoding a member of NAD+-dependent histone deacetylase (HDAC), was deleted to evaluate its regulation on the production of Monascus azaphilone pigments (MonAzPs) and mycotoxin, as well as the developmental process in Monascusruber. METHODS AND RESULTS: Agrobacterium tumefaciens-mediated transformation was applied in this study to generate the Mrhst4 null strain. Mrhst4-deleted strain did not display obvious differences in the sexual and asexual reproduction, colonial morphology, and micro-morphology. UV-Vis scan and UPLC detection showed that disruption of Mrhst4 significantly increased the MonAzPs yields, and citrinin content was dramatically enhanced during the tested period. RT-qPCR results showed that the absence of Mrhst4 significantly increased the relative expression of citrinin biosynthetic pathway genes including pksCT, mrl1, mrl2, mrl4, mrl6, and mrl7. The Western blot assay suggested that deletion of Mrhst4 could significantly elevate the acetylation levels of H3K4, H3K9, H3K18, H3K56, and H4K12, but attenuated the lysine acetylation modification of H4Pan, H4K8, and H4K16. CONCLUSION: MrHst4 is an important regulator involved in secondary metabolism in Monascus ruber. In particular, MrHst4 plays a pivotal role in regulation of citrinin production.

Overexpression of MrEsa1 accelerated growth, increased ascospores yield, and the polyketide production in Monascus ruber.

Esa1 has been proven to be an important histone acetyltransferase involved in the regulation of growth and metabolism. Monascus spp. with nearly 2000 years of edible history in East Asian countries can produce a variety of polyketides. It is unknown whether Esa1 plays a regulatory role in Monascus spp. In this study, we isolated the homology of histone acetyltransferase Esa1 (named MrEsa1) and constructed a mresa1-overexpressed strain. Western blot experiments showed that MrEsa1 hyperacetylated at K4 and K9 of the H3 subunit in Monascus ruber. Overexpression of mresa1 led to the larger colony diameter and increased dry cell mass; meanwhile, the conidia germination rate was significantly accelerated in the mresa1-overexpressed strain before 4 h, and the number of ascospores in the mresa1-overexpressed strain was significantly higher than that in WT. In addition, the Monascus azaphilone pigments (MonAzPs) and citrinin production of the mresa1-overexpressed strain were 1.7 and 2.4 times more than those of WT, respectively. Reverse transcription-quantitative polymerase chain reaction experiment suggested that mrpigB, mrpigH, mrpigJ, and mrpigK, involved in MonAzPs synthesis, and pksCT, mrl3, and mrl7, involved in citrinin synthesis, were upregulated in mresa1-overexpressed strain. This study provides important insights into the effect of MrEsa1 on the developmental process and the production of secondary metabolites in Monascus spp.

Histone deacetylase MrHos3 negatively regulates the production of citrinin and pigments in Monascus ruber.

Monascus spp. can produce a variety of beneficial metabolites widely used in food and pharmaceutical industries. However, some Monascus species contain the complete gene cluster responsible for citrinin biosynthesis, which raises our concerns about the safety of their fermented products. In this study, the gene Mrhos3, encoding histone deacetylase (HDAC), was deleted to evaluate its effects on the production of mycotoxin (citrinin) and the edible pigments as well as the developmental process of Monascus ruber M7. The results showed that absence of Mrhos3 caused an enhancement of citrinin content by 105.1%, 82.4%, 111.9%, and 95.7% at the 5th, 7th, 9th, and 11th day, respectively. Furthermore, deletion of Mrhos3 increased the relative expression of citrinin biosynthetic pathway genes including pksCT, mrl1, mrl2, mrl4, mrl6, and mrl7. In addition, deletion of Mrhos3 led to an increase in total pigment content and six classic pigment components. Western blot results revealed that deletion of Mrhos3 could significantly elevate the acetylation level of H3K9, H4K12, H3K18, and total protein. This study provides an important insight into the effects of hos3 gene on the secondary metabolites production in filamentous fungi.

Mrada3 is required for sexual reproduction and secondary metabolite production in industrial fungi Monascus strain.

AIMS: Monascus spp. are valuable industrial fungi for producing beneficial compounds. Because sporulation is often coupled with the production of secondary metabolites, the current study was performed to investigate how Mrada3 regulated asexual and sexual development and the production of edible pigments and mycotoxin. METHODS AND RESULTS: The functional characteristics of Mrada3 were identified by gene deletion and overexpression in Monascus ruber M7 (the wild-type, WT). The results revealed that the ΔMrada3 strain aborted sexual development, but it produced many more conidia than WT. RNA-seq data showed that the deletion of Mrada3 altered the expression levels of partial genes involved in sexual and asexual development. In addition, the deletion of Mrada3 also resulted in slower growth, lower pigment production and increased citrinin yield during the late period. For the Mrada3-overexpressed strain, the number of ascospores and pigment content were significantly higher than those of WT, but citrinin was slightly lower than that of WT. CONCLUSIONS: The Mrada3 gene plays a vital role in the sporulation development and secondary metabolism of Monascus species. SIGNIFICANCE AND IMPACT OF THE STUDY: Mrada3 is first identified as an essential regulator for sexual development in Monascus species, enriching the regulatory knowledge of sexual development in filamentous fungi.

Loss of HP1 causes depletion of H3K27me3 from facultative heterochromatin and gain of H3K27me2 at constitutive heterochromatin.

Methylated lysine 27 on histone H3 (H3K27me) marks repressed "facultative heterochromatin," including developmentally regulated genes in plants and animals. The mechanisms responsible for localization of H3K27me are largely unknown, perhaps in part because of the complexity of epigenetic regulatory networks. We used a relatively simple model organism bearing both facultative and constitutive heterochromatin, Neurospora crassa, to explore possible interactions between elements of heterochromatin. In higher eukaryotes, reductions of H3K9me3 and DNA methylation in constitutive heterochromatin have been variously reported to cause redistribution of H3K27me3. In Neurospora, we found that elimination of any member of the DCDC H3K9 methylation complex caused massive changes in the distribution of H3K27me; regions of facultative heterochromatin lost H3K27me3, while regions that are normally marked by H3K9me3 became methylated at H3K27. Elimination of DNA methylation had no obvious effect on the distribution of H3K27me. Elimination of HP1, which "reads" H3K9me3, also caused major changes in the distribution of H3K27me, indicating that HP1 is important for normal localization of facultative heterochromatin. Because loss of HP1 caused redistribution of H3K27me2/3, but not H3K9me3, these normally nonoverlapping marks became superimposed. Indeed, mass spectrometry revealed substantial cohabitation of H3K9me3 and H3K27me2 on H3 molecules from an hpo strain. Loss of H3K27me machinery (e.g., the methyltransferase SET-7) did not impact constitutive heterochromatin but partially rescued the slow growth of the DCDC mutants, suggesting that the poor growth of these mutants is partly attributable to ectopic H3K27me. Altogether, our findings with Neurospora clarify interactions of facultative and constitutive heterochromatin in eukaryotes.

Effects of an alternative oxidase gene on conidia viability under external stresses in Monascus ruber M7.

Monascus species can produce natural edible pigments and many other bioactive metabolites. In this study, mraox gene (Monascus ruber alternative oxidase) was isolated, sequenced, and replaced in order to investigate the function in resistance of conidia to stressful conditions. The derived protein of the mraox gene consisted of 350 amino acids with a conserved ferritin-like diiron-binding domain at the C-terminus, sharing a high homolog with alternative oxidase proteins in other filamentous fungi. Deletion of mraox gene repressed the conidia germination rate (CGR) when conidia were exposed to H2 O2 , high temperature (40 and 50 °C) and alkerline buffer (pH8.0), but CGR of mraox-deleted strain was not decreased when the conidia were treated with NaCl, acid buffer (citric acid-dibasic sodium phosphate buffer, pH3) compared to that of the wild-type strain, suggesting that mraox gene is partially responsible for the resistance of conidia to stressful conditions in M. ruber.

Cloning, expression and characterization of a novel cold-active and organic solvent-tolerant esterase from Monascus ruber M7.

Cold active esterases are a class of important biocatalysts that exhibit high activity at low temperatures. In this study, a search for putative cold-active esterase encoding genes from Monascus ruber M7 was performed. A cold-active esterase, named Lip10, was isolated, cloned, purified, and characterized. Amino acid sequence analysis reveals that Lip10 contained a conserved sequence motif Gly(173)-Xaa-Ser(175)-Xaa-Gly(177) that is also present in the majority of esterases and lipases. Phylogenetic analysis indicated that Lip10 was a novel microbial esterase. The lip10 gene was cloned and heterologously expressed in Escherichia coli BL21(DE3), resulting in the expression of an active and soluble protein that constituted 40 % of the total cell protein content. Lip10 maintained almost 50 % of its maximal activity at 4-10 °C, with optimal activity at 40 °C. Furthermore, Lip10 retained 184-216 % of its original activity, after incubation in 50 % (v/v) hydrophobic organic solvents for 24 h. The enzyme also exhibited high activity under alkaline conditions and good tolerance to metal ions in the reaction mixture. These results indicate that Lip10 may have potential uses in chemical synthesis and food processing industrial applications as an esterase.

Identification and role analysis of an intermediate produced by a polygenic mutant of Monascus pigments cluster in Monascus ruber M7.

Monascus pigments (Mps) are a group of azaphilonic secondary metabolites produced by Monascus spp. via a polyketide pathway. A mutant deleted an about 30 kb region of Mps gene cluster from Monascus ruber M7 was isolated previously, which produces a high amount of a light yellow pigment. The current study revealed that the mutant named ΔMpigJ-R lost proximate eight genes of the Mps gene cluster in M. ruber M7 through genetic analysis at DNA and RNA levels. The produced light yellow material was identified as a benzaldehyde derivative named as 6-(4-hydroxy-2-oxopentyl)-3-methyl-2, 4-dioxocyclohexane carb-aldehyde (M7PKS-1) by FT-IR, NMR, and MS. The sodium acetate-1-(13)C feeding experiment indicated that M7PKS-1 was a product produced from polyketide pathway. Finally, the feeding of M7PKS-1 helped to induce and regain Mps production of the mutants (ΔMpigA and ΔMpigE) which were previously unable to biosynthesize Mps and proved that M7PKS-1 was an initial intermediate of Mps. The results in this study provide a line of action to unveil Monascus pigments biosynthesis pathway.

mrskn7, a putative response regulator gene of Monascus ruber M7, is involved in oxidative stress response, development, and mycotoxin production.

Skn7, a response regulator (RR), is associated with oxidative stress adaptation, hypo-osmotic stress response, fungicide sensitivity, cell wall biosynthesis, cell cycle regulation, sexual mating, and sporulation in many filamentous fungi and yeasts. In this study a Skn7-like protein gene mrskn7 (Monascus ruber skn7) was isolated, sequenced, and disrupted to investigate its function in M. ruber Bioinformatics predicted that the deduced protein encoded by mrskn7 contained the conserved DNA-binding and signal-receiver domains similar to the Skn7-like protein structure in other filamentous fungi. The Δmrskn7 strain produced fewer conidia and less mycotoxin, demonstrated increased sensitivity to peroxide but the same level of osmotic resistance to NaCl and glycerol with the wild-type. Additionally, cleistothecia observed at different time point showed a different morphology between the wild-type and the Δmrskn7 strain, suggesting the involvement of mrskn7 in sexual development of M. ruber These results indicated that mrskn7 plays important roles in asexual and sexual development, the production of mycotoxin as well as regulation of oxidative stress signal in M. ruber.

Overview on mechanisms of acetic acid resistance in acetic acid bacteria.

Acetic acid bacteria (AAB) are a group of gram-negative or gram-variable bacteria which possess an obligate aerobic property with oxygen as the terminal electron acceptor, meanwhile transform ethanol and sugar to corresponding aldehydes, ketones and organic acids. Since the first genus Acetobacter of AAB was established in 1898, 16 AAB genera have been recorded so far. As the main producer of a world-wide condiment, vinegar, AAB have evolved an elegant adaptive system that enables them to survive and produce a high concentration of acetic acid. Some researches and reviews focused on mechanisms of acid resistance in enteric bacteria and made the mechanisms thoroughly understood, while a few investigations did in AAB. As the related technologies with proteome, transcriptome and genome were rapidly developed and applied to AAB research, some plausible mechanisms conferring acetic acid resistance in some AAB strains have been published. In this review, the related mechanisms of AAB against acetic acid with acetic acid assimilation, transportation systems, cell morphology and membrane compositions, adaptation response, and fermentation conditions will be described. Finally, a framework for future research for anti-acid AAB will be provided.

Insights into Monascus biology at the genetic level.

The genus of Monascus was nominated by van Tieghem in 1884, but its fermented product-red mold rice (RMR), namely red yeast rice, has been used as folk medicines, food colorants, and fermentation starters for more than thousands of years in oriental countries. Nowadays, RMR is widely developed as food supplements around the world due to its functional compounds such as monacolin K (MK, also called lovastatin) and γ-aminobutyric acid. But the usage of RMR also incurs controversy resulting from contamination of citrinin (a kind of mycotoxin) produced by some Monascus strains. In the past decade, it has made great progress to Monascus spp. at the genetic level with the application of molecular biology techniques to restrain the citrinin production and increase the yields of MK and pigment in RMR, as well as aid Monascus classification and phylogenesis. Up to now, hundreds of papers about Monascus molecular biology (MMB) have been published in the international primary journals. However, to our knowledge, there is no MMB review issued until now. In this review, current understanding of Monascus spp. from the view of molecular biology will be covered and insights into research areas that need to be further investigated will also be discussed.

MpigE, a gene involved in pigment biosynthesis in Monascus ruber M7.

Monascus pigments (MPs) have been used as food colorants for several centuries in Asian countries. However, MP biosynthesis pathway is still a controversy, and only few related genes have been reported. In this study, the function of MpigE, a gene involved in MP biosynthesis in Monascus ruber M7, was analyzed. The results revealed that the disruption, complementation, and overexpression of MpigE in M. ruber M7 had very little effects on the growth and phenotypes except MPs. The MpigE deletion strain (∆MpigE) just yielded four kinds of yellow MPs and very little red pigments, while the wild-type strain M. ruber M7 produced a MP complex mixture including three (orange, red, and yellow) categories of MP compounds. Two of the four yellow MPs produced by ∆MpigE were the same as those yielded by M. ruber M7. The MpigE complementation strain (∆MpigE::MpigE) recovered the ability to generate orange and red MPs as M. ruber M7. The MP types produced by the MpigE overexpression strain (M7::PtrpC-MpigE) were consistent with those of M. ruber M7, while the color value was about 1.3-fold as that of M. ruber M7 (3,129 U/g red kojic). For the production of citrinin, the disruption of MpigE almost had no influence on the strain, whereas the overexpression of MpigE made citrinin decrease drastically in YES fermentation. This work will make a contribution to the study on the biosynthesis pathway of MPs in M. ruber.

A novel phagomagnetic separation-ATP bioluminescence (PhMS-BL) for rapid and sensitive detection of viable Vibrio parahaemolyticus in aquatic product.

Detection of viable Vibrio parahaemolyticus (V. parahaemolyticus) is a major challenge due to its significant risk to food safety and human health. Herein, we developed a phagomagnetic separation-ATP bioluminescence (PhMS-BL) assay based on phage VPHZ6 for rapid and sensitive detection of viable V. parahaemolyticus. Phage as a recognition element was coupled to magnetic beads to capture and enrich V. parahaemolyticus, shortening detection time and improving method sensitivity. The intracellular ATP released by chemical lysis using CTAB was quantified using firefly fluorescein-adenosine triphosphate bioluminescence system to detect viable bacteria. So, PhMS-BL method was able to detect V. parahaemolyticus in a linear range of 2.3 × 102 to 1.3 × 107 CFU mL-1, with a detection limit of 78 CFU mL-1 within 15 min. It is successfully applied to detect V. parahaemolyticus in spiked lake water, lobster tail meat, and clam meat. The developed detection strategy can rapidly and sensitively detect viable V. parahaemolyticus in food matrixes.

Histone lysine methyltransferases MpDot1 and MpSet9 are involved in the production of lovastatin and MonAzPs by histone crosstalk modification.

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.

Exploring the Subcellular Localization of Monascus Pigments Biosynthases: Preliminary Unraveling of the Compartmentalization Mechanism.

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.

New Horizons of Covalent Complex of Plant-Derived Recombinant Human Lactoferrin (OsrhLF) Combined with Different Polyphenols: Formation, Physicochemical Properties, and Gastrointestinal Fate.

Four typical dietary polyphenols ((-)-epigallocatechin gallate (EGCG), quinic acid (QA), caffeic acid (CA), and ferulic acid (FA)) were covalently prepared with rice recombinant human lactoferrin (OsrhLF) and bovine lactoferrin (bLF), and their structure and physicochemical properties were investigated, different lycopene emulsions were made by ultrasonic emulsification to analyze gastrointestinal fate. The results indicated that the covalent modification polyphenols changed the secondary/tertiary structure of LF, significantly improving the surface hydrophilicity, thermal stability, and antioxidant activity of LF. Compared with the bLF group, the OsrhLF group was more hydrophilic and the thermal denaturation temperature of the OsrhLF-CA reached 104.4 °C. LF-polyphenol emulsions significantly enhanced the photochemical stability and bioavailability of lycopene and achieved effective encapsulation and protection of lycopene compared to free lycopene, and the OsrhLF-EGCG reached 58.94% lycopene bioavailability. In short, OsrhLF does not differ much from bLF in terms of physicochemical properties and has a strong potential in the field of dietary supplements.

Insight into selenium biofortification and the selenite metabolic mechanism of Monascus ruber M7.

Monascus species are functional fermentation fungi with great potential for selenium (Se) supplementation. This study investigated the effects of Se bio-fortification on the growth, morphology, and biosynthesis of Monascus ruber M7. The results demonstrated a significant increase in the yield of orange and red Monascus pigments (MPs) in red yeast rice (RYR) by 38.52% and 36.57%, respectively, under 20 µg/mL of selenite pressure. Meanwhile, the production of citrinin (CIT), a mycotoxin, decreased from 244.47 µg/g to 175.01 µg/g. Transcriptome analysis revealed significant upregulation of twelve genes involved in MPs biosynthesis, specifically MpigE, MpigF, and MpigN, and downregulation of four genes (mrr3, mrr4, mrr7, and mrr8) associated with CIT biosynthesis. Additionally, three genes encoding cysteine synthase cysK (Log2FC = 1.6), methionine synthase metH (Log2FC = 2.2), and methionyl-tRNA synthetase metG (Log2FC = 1.8) in selenocompound metabolism showed significantly upregulated. These findings provide insights into Se biotransformation and metabolism in filamentous fungi.

An Innovative and Efficient Fluorescent Detection Technique for Salmonella in Animal-Derived Foods Using the CRISPR/Cas12a-HCR System Combined with PCR/RAA.

Here, we present a method for Salmonella detection using clustered regularly interspaced short palindromic repeats associated with the CRISPR-associated protein 12a-hybridization chain reaction (CRISPR/Cas12a-HCR) system combined with polymerase chain reaction/recombinase-assisted amplification (PCR/RAA) technology. The approach relies on the Salmonella invA gene as a biorecognition element and its amplification through PCR and RAA. In the presence of the target gene, Cas12a, guided by crRNA, recognizes and cleaves the amplification product, initiating the HCR. Fluorescently labeled single-stranded DNA (ssDNA) H1 and H2 were introduced, and the Salmonella concentration was determined based on the fluorescence intensity from the triggered HCR. Both assays demonstrate high specificity, sensitivity, simplicity, and rapidity. The detection range was 2 × 101-2 × 109 CFU/mL, with an LOD of 20 CFU/mL, and the entire process enabled specific and rapid Salmonella detection within 85-105 min. Field-incurred spiked recovery tests were conducted in mutton and beef samples using both assays, demonstrating satisfactory recovery and accuracy in animal-derived foods. By combining CRISPR/Cas12a with hybridization chain reaction technology, this study presents a rapid and sensitive Salmonella detection method that is crucial for identifying pathogenic bacteria and monitoring food safety.

Evolution of complete proteomes: guanine-cytosine pressure, phylogeny and environmental influences blend the proteomic architecture.

BACKGROUND: Guanine-cytosine (GC) composition is an important feature of genomes. Likewise, amino acid composition is a distinct, but less valued, feature of proteomes. A major concern is that it is not clear what valuable information can be acquired from amino acid composition data. To address this concern, in-depth analyses of the amino acid composition of the complete proteomes from 63 archaea, 270 bacteria, and 128 eukaryotes were performed. RESULTS: Principal component analysis of the amino acid matrices showed that the main contributors to proteomic architecture were genomic GC variation, phylogeny, and environmental influences. GC pressure drove positive selection on Ala, Arg, Gly, Pro, Trp, and Val, and adverse selection on Asn, Lys, Ile, Phe, and Tyr. The physico-chemical framework of the complete proteomes withstood GC pressure by frequency complementation of GC-dependent amino acid pairs with similar physico-chemical properties. Gln, His, Ser, and Val were responsible for phylogeny and their constituted components could differentiate archaea, bacteria, and eukaryotes. Environmental niche was also a significant factor in determining proteomic architecture, especially for archaea for which the main amino acids were Cys, Leu, and Thr. In archaea, hyperthermophiles, acidophiles, mesophiles, psychrophiles, and halophiles gathered successively along the environment-based principal component. Concordance between proteomic architecture and the genetic code was also related closely to genomic GC content, phylogeny, and lifestyles. CONCLUSIONS: Large-scale analyses of the complete proteomes of a wide range of organisms suggested that amino acid composition retained the trace of GC variation, phylogeny, and environmental influences during evolution. The findings from this study will help in the development of a global understanding of proteome evolution, and even biological evolution.