Molecular cloning and characterization of an ML-236B (compactin) biosynthetic gene cluster in Penicillium citrinum

Cloning of genes encoding polyketide synthases (PKSs) has allowed us to identify a gene cluster for ML-236B biosynthesis in Penicillium citrinum. Like lovastatin, which is produced by Aspergillus terreus, ML-236B (compactin) inhibits the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. Genomic sequencing and Northern analysis showed that nine predicted genes for ML-236B biosynthesis were located within a 38-kb region and were transcribed when ML-236B was produced. The predicted amino acid sequences encoded by these nine genes, designated mlcA- mlcH and mlcR, were similar to those encoded by the genes for lovastatin synthesis, and were therefore assumed to be involved either directly or indirectly in ML-236B biosynthesis. Targeted disruption experiments provided evidence that two PKS genes in the cluster, mlcA and mlcB, are required for the biosynthesis of the nonaketide and the diketide moieties, respectively, of ML-236B, suggesting that the gene cluster as a whole is responsible for ML-236B biosynthesis in P. citrinum. Bioconversion of some of the predicted intermediates by an mlcA-disrupted mutant was also investigated in order to analyze the ML-236B biosynthetic pathway. The molecular organization of the gene cluster and proposed functions for the ML-236B biosynthetic genes in P. citrinum are described.     

Effect of increased dosage of the ML-236B (compactin) biosynthetic gene cluster on ML-236B production in<Emphasis Type="Italic"> Penicillium citrinum</Emphasis>

The gene cluster responsible for ML-236B (compactin) biosynthesis has recently been characterized from P. citrinum No. 41520. Here, we describe how the ML-236B-producing strain was improved using a cosmid-mediated recombination technique. The introduction of the cosmid pML48, which contains seven of the nine ML-236B biosynthetic genes, into P. citrinum No. 41520 resulted in transformants which produced increased amounts of ML-236B. Southern analysis showed that pML48 had been incorporated by a homologous recombination event, and all high producers possessed two copies of each of the seven genes, mlcA- mlcF and mlcR, suggesting that increased dosage of the biosynthetic gene cluster was responsible for the enhanced production of ML-236B. On the other hand, various kinds of mutants with decreased titers of ML-236B were also obtained. Characterization of one such mutant, designated as T48.28, which was more sensitive to ML-236B than the parental strain, suggested that one of the ML-236B biosynthetic genes, mlcD, which encodes a putative HMG-CoA reductase, was involved in conferring resistance to ML-236B.     

Improvement of compactin (ML-236B) production by genetic engineering in compactin high-producing <Emphasis Type="Italic">Penicillium citrinum</Emphasis>

An increase in compactin (ML-236B) production was achieved by introducing a whole compactin biosynthetic gene cluster or the regulatory gene mlcR into compactin high-producing Penicillium citrinum. In the previous report, we introduced mlcR encoding the positive regulator of compactin biosynthetic genes into compactin high-producing strain no. 41520, and most of the transformants produced higher amounts of compactin. Here, we characterize one of the resulting high producers (strain TIR-35, which produced 50% more compactin) and reveal that TIR-35 contained five copies of mlcR and that early, enhanced expression of mlcR caused compactin overproduction. Similarly, the introduction of mlcR into strain T48.19, which was created previously from strain no. 41520 by introducing a partial compactin biosynthetic gene cluster, enhanced compactin production further. Our results indicated that genetic engineering is an effective tool to improve compactin production, even in compactin high producers.     

Microbial Transformation of ML-236B (Compactin) to M3, a Mammalian Metabolite of ML-236B

In a previous study, we synthesized a novel inhibitor of ceramide kinase, K1. In this study, we determined that inhibition by K1 is non-competitive and that four intact six-membered rings are important to the inhibitory activity. Furthermore, we identified an effective in vivo concentration for K1, at which it did not influence any cellular lipid synthesis other than that of ceramide 1-phosphate (C1P) using RBL-2H3 cells, and found that K1 suppressed the activation of mast cells.     

A chemically-defined medium for production of compactin by Penicillium citrinum

A mutant strain of Penicillium citrinum grown in a chemically-defined production medium, yielded 145 mg compactin l^–1. The medium also facilitated spectrophotometric analysis of compactin. Addition of KH₂PO₄in the production medium did not increase the compactin production, while addition of a surfactant, Tween 80, increased compactin to 175 mg l^–1. Inoculation with 10⁷ spores ml^–1 and initial pH of 6.5–7 were the most suitable for compactin production.     

Cellulase production in a new mutant strain of Penicillium decumbens ML-017 by solid state fermentation with rice bran

To produce cellulolytic enzyme efficiently, Penicillium decumbens strain L-06 was used to prepare mutants with ethyl methane sulfonate (EMS) and UV-irradiation. A mutant strain ML-017 is shown to have a higher cellulase activity than others. Box-Behnken's design (BBD) and response surface methodology (RSM) were adopted to optimize the conditions of cellulase (filter paper activity, FPA) production in strain ML-017 by solid-state fermentation (SSF) with rice bran as the substrate. And the result shows that the initial pH, moisture content and culture temperature all have significant effect on the production of cellulase. The optimized condition shall be initial pH 5.7, moisture content 72% and culture temperature 30°C. The maximum cellulase (FPA) production was obtained under the optimized condition, which is 5.76 IU g(-1), increased by 44.12% to its original strain. It corresponded well with the calculated results (5.15 IU g(-1)) by model prediction. The result shows that both BBD and RSM are the cellulase optimization methods with good prospects.     

Double-stranded ribonucleic acid in viruses of Penicillium citrinum.

The nucleic acid content of two viral populations in a strain of Penicillium citrinum is shown to be double-stranded ribonucleic acid, resolved through polyacrylamide gel electrophoresis into 10 size groups ranging from 1.17 to 3.98 megadaltons.     

Double-stranded ribonucleic acid in viruses of Penicillium citrinum.

The nucleic acid content of two viral populations in a strain of Penicillium citrinum is shown to be double-stranded ribonucleic acid, resolved through polyacrylamide gel electrophoresis into 10 size groups ranging from 1.17 to 3.98 megadaltons.     

Energetics of growth and penicillin production in a high-producing strain of Penicillium chrysogenum

The results of a large number of carbon-limited chemostat cultures of Penicillium chrysogenum carried out on glucose, ethanol, and acetate as the growth limiting substrate have been used to obtain an estimation of the adenosine triphosphate (ATP) costs for mycelium growth, penicillin production, and maintenance and the overall stoichiometry of oxidative phosphorylation of the fungus. It was found that penicillin production was accompanied by a significant additional energy drain (73 mol of ATP per mole of penicillin-G) from primary metabolism. This finding has been confirmed in independent experiments and has been shown to result in a significantly lower estimate for the maximum theoretical yield of penicillin-G on the carbon source.     

过表达全局调控因子LaeA对橘青霉美伐他汀的合成及产孢的影响

【目的】研究橘青霉中全局性调控因子Lae A过表达对美伐他汀生物合成过程及产孢的影响。【方法】同源克隆法从橘青霉中克隆获得lae A基因,构建p Gi HTGi-lae A载体,经农杆菌介导转化转入橘青霉,利用hygromycin基因进行阳性克隆子的PCR筛选。使用HPLC方法比较PGA发酵液中橘青霉野生型菌株WT和Lae A过表达菌株OE::lae A的美伐他汀产量差异。血球计数法进行孢子产量比较。荧光定量PCR方法分析WT和OE::lae A中美伐他汀合成基因簇表达量。【结果】成功构建p Gi HTGi-lae A载体,并获得Lae A过表达橘青霉菌株(OE::lae A)。OE::lae A中,lae A基因表达量较WT增加29%,mlc B表达量较WT增加72%,mlcR表达量增加153%,孢子产量由(2.38±0.24)×107/cm2减少到(1.40±0.11)×107/cm2,美伐他汀产量从(35.77±4.63)mg/L提高到(201.46±9.98)mg/L。【结论】在橘青霉中过表达Lae A可能通过提高mlcR和mlc B基因的转录来增加美伐他汀的产量,同时过表达lae A基因不利于孢子的形成。这些结果为美伐他汀生物合成的全局性调控和高产菌株的开发提供基础。     

Double-Stranded Ribonucleic Acid in Viruses of Penicillium citrinum

[This corrects the article on p. 811 in vol. 34.].     

Presence of Two Virus-Like Particles in Penicillium citrinum

Two icosahedral virus-like particles (28 and 19 nm in diameter, respectively) have been detected in sporogenic and asporogenic segregants of a strain of Penicillium citrinum. The distribution of the two particles differed among the two segregants.     

Molecular basis for mycophenolic acid biosynthesis in Penicillium brevicompactum

Mycophenolic acid (MPA) is the active ingredient in the increasingly important immunosuppressive pharmaceuticals CellCept (Roche) and Myfortic (Novartis). Despite the long history of MPA, the molecular basis for its biosynthesis has remained enigmatic. Here we report the discovery of a polyketide synthase (PKS), MpaC, which we successfully characterized and identified as responsible for MPA production in Penicillium brevicompactum. mpaC resides in what most likely is a 25-kb gene cluster in the genome of Penicillium brevicompactum. The gene cluster was successfully localized by targeting putative resistance genes, in this case an additional copy of the gene encoding IMP dehydrogenase (IMPDH). We report the cloning, sequencing, and the functional characterization of the MPA biosynthesis gene cluster by deletion of the polyketide synthase gene mpaC of P. brevicompactum and bioinformatic analyses. As expected, the gene deletion completely abolished MPA production as well as production of several other metabolites derived from the MPA biosynthesis pathway of P. brevicompactum. Our work sets the stage for engineering the production of MPA and analogues through metabolic engineering.     

Biosynthesis of pectinlyases in Penicillium adametzii,P. citrinum and P. janthinellum

Pectinlyase complexes of Penicillium adametzii, P. citrinum and P. janthinellum occur as multiple molecular forms distinguished by their biosynthetic control. AMP is involved in derepression of pectinlyase formation.The authors are with the Institute of Microbiology, Belarus Academy of Sciences, Zhodinskaya 2, 220141, Minsk, Republic of Belarus.