State Key Laboratory of Mycology

The emerging human fungal pathogen Candida auris has been recognized as a multidrug resistant species and is associated with high mortality. This fungus was first described in Japan in 2009 and has been reported in at least 18 countries on five continents. In this study, we report the first isolate of C. auris from the bronchoalveolar lavage fluid (BALF) of a hospitalized woman in China. Interestingly, this isolate is susceptible to all tested antifungals including amphotericin B, fluconazole, and caspofungin. Copper sulfate (CuSO4) also has a potent inhibitory effect on the growth of this fungus. Under different culture conditions, C. auris exhibits multiple morphological phenotypes including round-to-ovoid, elongated, and pseudohyphal-like forms. High concentrations of sodium chloride induce the formation of a pseudohyphal-like form. We further demonstrate that C. auris is much less virulent than Candida albicans in both mouse systemic and invertebrate Galleria mellonella models.

The human commensal fungus Candida albicans can cause not only superficial infections, but also life-threatening disease in immunocompromised individuals. C. albicans can grow in several morphological forms. The ability to switch between different phenotypic forms has been thought to contribute to its virulence. The yeast-filamentous growth transition and white-opaque switching represent two typical morphological switching systems, which have been intensively studied in C. albicans. The interplay between environmental factors and genes determines the morphology of C. albicans. This review focuses on the regulation of phenotypic changes in this pathogenic organism by external environmental cues and internal genes.

This review covers diverse transcriptional regulators for the activation of secondary metabolism and novel natural product discovery in fungi.

A total of 124 Pestalotiopsis-like isolates associated with symptomatic and asymptomatic tissues of Camellia sinensis and other Camellia spp. from eight provinces in China were investigated. Based on single- and multi-locus (ITS, TEF, TUB2) phylogenies, as well as morphological characters, host associations and geographical distributions, they were classified into at least 19 species in three genera, i.e. Neopestalotiopsis, Pestalotiopsis and Pseudopestalotiopsis. Eight novel species in Pestalotiopsis and three novel species in Pseudopestalotiopsis were described. Our data suggested that the currently widely used loci in Pestalotiopsis-like genera do not consistently provide stable and sufficient resolution tree topologies, especially for Neopestalotiopsis. Moreover, the number, branch pattern and length of the conidial basal appendages were revealed to be phylogenetically informative characters in Pestalotiopsis.

In a preliminary analysis, 21 Colletotrichum strains with large conidia preserved in the CBS culture collection clustered with a recently described species, C. gigasporum, forming a clade distinct from other currently known Colletotrichum species complexes. Multi-locus phylogenetic analyses (ITS, ACT, TUB2, CHS-1, GAPDH) as well as each of the single-locus analyses resolved seven distinct species, one of them being C. gigasporum. Colletotrichum gigasporum and its close allies thus constitute a previously unknown species complex with shared morphological features. Five of the seven species accepted in the C. gigasporum species complex are described here as novel species, namely C. arxii, C. magnisporum, C. pseudomajus, C. radicis and C. vietnamense. A species represented by a single sterile strain, namely CBS 159.50, was not described as novel species, and is treated as Colletotrichum sp. CBS 159.50. Furthermore, C. thailandicum is reduced to synonymy with C. gigasporum.

Lactic acid (LA) is an important organic acid with broad industrial applications. Considered as an environmentally friendly alternative to petroleum-based plastic with a wide range of applications, polylactic acid has generated a great deal of interest and therefore the demand for optically pure l- or d-lactic acid has increased accordingly. Microbial fermentation is the industrial route for LA production. LA bacteria and certain genetic engineering bacteria are widely used for LA production. Although some fungi, such as Saccharomyces cerevisiae, are not natural LA producers, they have recently received increased attention for LA production because of their acid tolerance. The main challenge for LA bioproduction is the high cost of substrates. The development of LA production from cost-effective biomasses is a potential solution to reduce the cost of LA production. This review examined and discussed recent progress in optically pure l-lactic acid and optically pure d-lactic acid fermentation. The utilization of inexpensive substrates is also focused on. Additionally, for PLA production, a complete biological process by one-step fermentation from renewable resources is also currently being developed by metabolically engineered bacteria. We also summarize the strategies and procedures for metabolically engineering microorganisms producing PLA. In addition, there exists some challenges to efficiently produce PLA, therefore strategies to overcome these challenges through metabolic engineering combined with enzyme engineering are also discussed.

Abstract Loss of heterozygosity (LOH) is observed during vegetative growth and reproduction of diploid genotypes through mitotic crossovers, aneuploidy caused by nondisjunction, and gene conversion. We aimed to test the role that LOH plays during adaptation of two highly heterozygous Saccharomyces cerevisiae genotypes to multiple environments over a short time span in the laboratory. We hypothesized that adaptation would be observed through parallel LOH events across replicate populations. Using genome resequencing of 70 clones, we found that LOH was widespread with 5.2 LOH events per clone after ∼500 generations. The most common mode of LOH was gene conversion (51%) followed by crossing over consistent with either break-induced replication or double Holliday junction resolution. There was no evidence that LOH involved nondisjunction of whole chromosomes. We observed parallel LOH in both an environment-specific and environment-independent manner. LOH largely involved recombining existing variation between the parental genotypes, but also was observed after de novo, presumably beneficial, mutations occurred in the presence of canavanine, a toxic analog of arginine. One highly parallel LOH event involved the ENA salt efflux pump locus on chromosome IV, which showed repeated LOH to the allele from the European parent, an allele originally derived by introgression from S. paradoxus. Using CRISPR-engineered LOH we showed that the fitness advantage provided by this single LOH event was 27%. Overall, we found extensive evidence that LOH could be adaptive and is likely to be a greater source of initial variation than de novo mutation for rapid evolution of diploid genotypes.

Summary The β‐nicotinamide mononucleotide (NMN) is a key intermediate of an essential coenzyme for cellular redox reactions, NAD. Administration of NMN is reported to improve various symptoms, such as diabetes and age‐related physiological decline. Thus, NMN is attracting much attention as a promising nutraceutical. Here, we engineered an Escherichia coli strain to produce NMN from cheap substrate nicotinamide (NAM) and glucose. The supply of in vivo precursor phosphoribosyl pyrophosphate (PRPP) and ATP was enhanced by strengthening the metabolic flux from glucose. A nicotinamide phosphoribosyltransferase with high activity was newly screened, which is the key enzyme for converting NAM to NMN with PRPP as cofactor. Notably, the E. coli endogenous protein YgcS, which function is primarily in the uptake of sugars, was firstly proven to be beneficial for NMN production in this study. Fine‐tuning regulation of ygc S gene expression in the engineered E. coli strain increased NMN production. Combined with process optimization of whole‐cell biocatalysts reaction, a final NMN titre of 496.2 mg l ‐1 was obtained.

Summary Mevalonate (MVA) pathway is the core for terpene and sterol biosynthesis, whose metabolic flux influences the synthesis efficiency of such compounds. Saccharomyces cerevisiae is an attractive chassis for the native active MVA pathway. Here, the truncated form of Enterococcus faecalis MvaE with only 3‐Hydroxy‐3‐methylglutaryl coenzyme A reductase (HMGR) activity was found to be the most effective enzyme for MVA pathway flux using squalene as the metabolic marker, resulting in 431‐fold and 9‐fold increases of squalene content in haploid and industrial yeast strains respectively. Furthermore, a positive correlation between MVA metabolic flux and β‐alanine metabolic activity was found based on a metabolomic analysis. An industrial strain SQ3‐4 with high MVA metabolic flux was constructed by combined engineering HMGR activity, NADPH regeneration, cytosolic acetyl‐CoA supply and β‐alanine metabolism. The strain was further evaluated as the chassis for terpenoids production. Strain SQ3‐4‐CPS generated from expressing β‐caryophyllene synthase in SQ3‐4 produced 11.86 ± 0.09 mg l −1 β‐caryophyllene, while strain SQ3‐5 resulted from down‐regulation of ERG1 in SQ3‐4 produced 408.88 ± 0.09 mg l −1 squalene in shake flask cultivations. Strain SQ3‐5 produced 4.94 g l −1 squalene in fed‐batch fermentation in cane molasses medium, indicating the promising potential for cost‐effective production of squalene.

BACKGROUND: 2-phenylethanol (2-PE) is an important aromatic compound with a lovely rose-like scent. Saccharomyces cerevisiae is a desirable microbe for 2-PE production but its natural yield is not high, and one or two crucial genes' over-expression in S. cerevisiae did not improve 2-PE greatly. RESULTS: A new metabolic module was established here, in which, permease Gap1p for L-phenylalanine transportation, catalytic enzymes Aro8p, Aro10p and Adh2p in Ehrlich pathway respectively responsible for transamination, decarboxylation and reduction were assembled, besides, glutamate dehydrogenase Gdh2p was harbored for re-supplying another substrate 2-oxoglutarate, relieving product glutamate repression and regenerating cofactor NADH. Due to different promoter strengths, GAP1, ARO8, ARO9, ARO10, ADH2 and GDH2 in the new modularized YS58(G1-A8-A10-A2)-GDH strain enhanced 11.6-, 15.4-, 3.6-, 17.7-, 12.4- and 7.5-folds respectively, and crucial enzyme activities of aromatic aminotransferases and phenylpyruvate decarboxylase were 4.8- and 7-folds respectively higher than that of the control. CONCLUSIONS: of 2-PE titer in 5-L fermenter reaching 95% of conversation ratio. Under fed-batch fermentation, 2-PE productivity at 24 h increased 29% than that of single-batch fermentation. Metabolic modularization with promoter strategy provides a new prospective for efficient 2-PE production.

The biosynthesis of diazobenzofluorene kinamycins requires a hitherto uncharacterized B-ring contraction. Via detailed genetic and enzymatic analyses we unambiguously characterized the conserved pairs of oxidases, AlpJ and AlpK homologs, as nature's machinery for benzofluorenone formation, which paves the way for the investigation of the following diazo assembly.

Summary Hazardous materials, such as heavy metals, are the major sources of health risk. Using genetically modified organisms (GMOs) to dispose heavy metals has the advantages of strong environmental compatibility and high efficiency. However, the biosecurity of GMOs used in the environment is a major concern. In this study, a self‐controlled genetic circuit was designed and carefully fine‐tuned for programmable expression in Pseudomonas putida KT2440, which is a widely used strain for environmental bioremediation. The cell behaviours were controlled by automatically sensing the variation of Hg 2+ concentration without any inducer requirement or manual interventions. More than 98% Hg 2+ was adsorbed by the engineered strain with a high cell recovery rate of 96% from waterbody. The remaining cells were killed by the suicide module after the mission was accomplished. The escape frequency of the engineered P . putida strain was lower than 10 −9 , which meets the recommendation of US NIH guideline for GMOs release (<10 −8 ). The same performance was achieved in a model experiment by using natural lake water with addition of Hg 2+ . The microbial diversity analysis further confirmed that the remediation process made little impact on the indigenous ecosystem. Thus, this study provides a practical method for environmental remediation by using GMOs.

The induction of fungal metabolites by fungal co-cultures grown on solid media was explored using multi-well co-cultures in 2 cm diameter Petri dishes. Fungi were grown in 12-well plates to easily and rapidly obtain the large number of replicates necessary for employing metabolomic approaches. Fungal culture using such a format accelerated the production of metabolites by several weeks compared with using the large-format 9 cm Petri dishes. This strategy was applied to a co-culture of a Fusarium and an Aspergillus strain. The metabolite composition of the cultures was assessed using ultra-high pressure liquid chromatography coupled to electrospray ionisation and time-of-flight mass spectrometry, followed by automated data mining. The de novo production of metabolites was dramatically increased by nutriment reduction. A time-series study of the induction of the fungal metabolites of interest over nine days revealed that they exhibited various induction patterns. The concentrations of most of the de novo induced metabolites increased over time. However, interesting patterns were observed, such as with the presence of some compounds only at certain time points. This result indicates the complexity and dynamic nature of fungal metabolism. The large-scale production of the compounds of interest was verified by co-culture in 15 cm Petri dishes; most of the induced metabolites of interest (16/18) were found to be produced as effectively as on a small scale, although not in the same time frames. Large-scale production is a practical solution for the future production, identification and biological evaluation of these metabolites.

A FAD-dependent oxidoreductase TdaR was responsible for α, β-disulfide formation in the biosynthesis of pretrichodermamide A. TdaR, together with its homologs AclT and GliT, catalysed not only α, α- but also α, β-disulfide formation in fungi.

Fusarins G1/G2 (1/2) and G3/G4 (3/4), two sets of interesting examples of diastereoisomers with substantially identical NMR data, were discovered from natural products. The reason was discussed and the generally applicable determinant conditions were proposed. The minimum interval for stereoclusters to be entirely segregated was also discussed.

Abstract Background Poly-γ-glutamic acid (γ-PGA) is a biopolymer and has various applications based on its biocompatibility, non-toxicity, and edibility. Low-molecular-weight (Mw)-γ-PGA has promising applications in agriculture and pharmaceuticals. It is traditionally produced by enzymatic hydrolysis. Cost-effective bioproduction of low-Mw-γ-PGA is essential for commercial application of γ-PGA. Results Bacillus subtilis 242 is a newly isolated low-Mw-γ-PGA-producing strain. To develop cost-effective production of γ-PGA using this newly isolated strain, cane molasses and corn steep liquor were used to produce γ-PGA. The concentration of cane molasses was optimized and 100 g/L cane molasses resulted in high γ-PGA production. The effects of yeast extract and corn steep liquor on γ-PGA yield were investigated. High concentration of γ-PGA was obtained in the medium with corn steep liquor. A concentration of 32.14 g/L γ-PGA was achieved in fed-batch fermentation, with a productivity of 0.67 g/L/h and a percentage yield ( g γ-PGA / g glutamate ) of 106.39%. The Mw of γ-PGA was 27.99 kDa. Conclusion This study demonstrated the potential application of B. subtilis 242 for cost-effective production of low-Mw-γ-PGA from cane molasses.

MOTIVATION: Computational protein sequence design has been widely applied in rational protein engineering and increasing the design accuracy and efficiency is highly desired. RESULTS: Here, we present ProDESIGN-LE, an accurate and efficient approach to protein sequence design. ProDESIGN-LE adopts a concise but informative representation of the residue's local environment and trains a transformer to learn the correlation between local environment of residues and their amino acid types. For a target backbone structure, ProDESIGN-LE uses the transformer to assign an appropriate residue type for each position based on its local environment within this structure, eventually acquiring a designed sequence with all residues fitting well with their local environments. We applied ProDESIGN-LE to design sequences for 68 naturally occurring and 129 hallucinated proteins within 20 s per protein on average. The designed proteins have their predicted structures perfectly resembling the target structures with a state-of-the-art average TM-score exceeding 0.80. We further experimentally validated ProDESIGN-LE by designing five sequences for an enzyme, chloramphenicol O-acetyltransferase type III (CAT III), and recombinantly expressing the proteins in Escherichia coli. Of these proteins, three exhibited excellent solubility, and one yielded monomeric species with circular dichroism spectra consistent with the natural CAT III protein. AVAILABILITY AND IMPLEMENTATION: The source code of ProDESIGN-LE is available at https://github.com/bigict/ProDESIGN-LE.

By disruption of LaeB, a global regulator recently characterized in Aspergillus nidulans, eight cryptic compounds in the mutant were identified, including seven polyketides and one NRPS-like product. Among the isolates, two phthalides and two dibenzo[1,4]dioxins are new compounds, revealing that the genetic manipulation of the global regulator represents a promising approach for the discovery of novel natural products in fungi.

Riboflavin (vitamin B2) is one of the essential vitamins that the human body needs to maintain normal metabolism. Its biosynthesis has become one of the successful models for gradual replacement of traditional chemical production routes. B. subtilis is characterized by its short fermentation time and high yield, which shows a huge competitive advantage in microbial fermentation for production of riboflavin. This review summarized the advancements of regulation on riboflavin production as well as the synthesis of two precursors of ribulose-5-phosphate riboflavin (Ru5P) and guanosine 5′-triphosphate (GTP) in B. subtilis. The different strategies to improve production of riboflavin by metabolic engineering were also reviewed.

, we show that two NRPSs, ApmA and ApmB, are sufficient for the synthesis of an amino acid ester, asperphenamate. Using the heterologous expression system, we identified that ApmA, with a reductase domain, rarely generates dipeptidyl alcohol. In contrast, ApmB was determined to not only catalyse inter-molecular ester bond formation but also accept the linear dipeptidyl precursor into the NRPS chain. The mechanism described here provides an approach for the synthesis of new small molecules with NRPS as the catalyst. Our study reveals for the first time a two-module NRPS system for the formation of amino acid esters in nature.