CefR modulates transporters of beta-lactam intermediates preventing the loss of penicillins to the broth and increases cephalosporin production in Acremonium chrysogenum

The Acremonium chrysogenum cephalosporin biosynthetic genes are divided in two different clusters. The central step of the biosynthetic pathway (epimerization of isopenicillin N to penicillin N) occurs in peroxisomes. We found in the “early” cephalosporin cluster a new ORF encoding a regulatory protein (CefR), containing a nuclear targeting signal and a “Fungal_trans” domain. Targeted inactivation of cefR delays expression of the cefEF gene, increases penicillin N secretion and decreases cephalosporin production. Overexpression of the cefR gene decreased (up to 60%) penicillin N secretion, saving precursors and resulting in increased cephalosporin C production. Northern blot analysis revealed that the CefR protein acts as a repressor of the exporter cefT and exerts a small stimulatory effect over the expression level of cefEF that explains the increased cephalosporin yields observed in transformants overexpressing cefR. In summary, we describe for the first time a modulator of beta-lactam intermediate transporters in A. chrysogenum.     

The NADP-dependent glutamate dehydrogenase gene from Penicillium chrysogenum and the construction of expression vectors for filamentous fungi

The gdhA gene encoding the NADP-dependent glutamate dehydrogenase activity from Penicillium chrysogenum has been isolated and characterized for its use in gene expression. The nucleotide sequence of a 2816-bp genomic fragment was determined, showing an open reading frame of 1600 bp interrupted by two introns, of 160 bp and 57 bp respectively, with fungal consensus splice-site junctions. The predicted amino acid sequence revealed a high degree of identity to glutamate dehydrogenase enzymes, especially to those from the fungi Aspergillus nidulans (82%) and Neurospora crassa (78%). The gdhA gene was found to be present in a single copy in the genome of several P. chrysogenum strains with different penicillin productivity. The use of the gdhA promoter for homologous and heterologous gene expression in fungi and Escherichia coli was analyzed. Heterologous gene expression was ascertained by the construction of gene fusions with the lacZ gene from E. coli and the bleomycin-resistance determinant (ble ^R) from Streptoalloteichus hindustanus. Homologous gene expression was shown through the use of the penicillin-biosynthetic genes pcbC and penDE from P. chrysogenum and the cephalosporin biosynthetic genes cefEF and cefG from Acremonium chrysogenum. Received: 2 November 1998 / Received revision: 15 January 1999 / Accepted: 5 March 1999     

Undefined xylose media extracted from biorefinery waste for enhanced and eco-friendly production of cephalosporin C by Acremonium chrysogenum M35

Xylose-rich undefined broth, extracted from the dilute acid pretreatment wastes of barley straw, serves as resourceful media for Acremonium chrysogenum M35 culture and production of cephalosporin C (CPC). Concentrating the extract with proper reprocessing enables to prepare various concentrations of xylose broth (2%–8%). The undefined xylose media were prepared for CPC production from A. chrysogenum M35 by the addition of other nutrients. Cell growth and CPC production were the most effective at 6% xylose and additional 2% glycerol, with maximum CPC production of 9.07 g/L after 6 days, which is higher production than that in defined media prepared with laboratory-level nutrients and reagents. Investigation of autotrophic and reverse trans-sulfuration pathways for cysteine synthesis, a limited element of three precursors for CPC synthesis, supports the enhanced CPC production in undefined media. Abundance of xylose ensures a maintained NADPH concentration required for sulfate reduction and synthesis of amino sulfide such as cysteine. Cystathionine-γ-lyase activity profiling indicated more efficient biosynthesis in undefined media than in other cultures use glycerol and glucose, and the biosynthesis pathway of CPC production by the cephalosporin gene cluster (i.e. pcbC and cefG genes) was investigated. The process using undefined xylose media was designed, and process simulation program confirmed our results.     

Chromosomal polymorphism of Acremonium chrysogenum strains producing cephalosporin C

Using pulse electrophoresis in controlled homogenous electric field we performed molecular karyotyping of cephalosporin C-producing industrial and laboratory strains of Acremonium chrysogenum. Differences in size of several chromosomes of high-producing strain CB26/8 compared to the wild-type strain ATCC 11550 were revealed. It was shown that chromosomal polymorphism in the high-producing strain was not associated with alteration of localization and copy number of cephalosporin C (CPC) biosynthesis and transport genes. A cluster of ??early?? CPC biosynthesis genes is located on chromosome VI (4.4 Mb); a cluster of the ??late genes??, on chromosome II (2.3 Mb). Both clusters are presented as a single copy per A. chrysogenum genome in the wild-type and in CB26/8 high-producing strains. Based on comparative analysis of laboratory and industrial CPC producers, a karyotype scheme for A. chrysogenum strains of various origins was designed.     

Expression of<Emphasis Type="Italic"> cefD2</Emphasis> and the conversion of isopenicillin N into penicillin N by the two-component epimerase system are rate-limiting steps in cephalosporin biosynthesis

The conversion of isopenicillin N into penicillin N in Acremonium chrysogenum is catalyzed by an epimerization system that involves an isopenicillin N-CoA synthethase and isopenicillin N-CoA epimerase, encoded by the genes cefD1 and cefD2. Several transformants containing two to seven additional copies of both genes were obtained. Four of these transformants (TMCD26, TMCD53, TMCD242 and TMCD474) showed two-fold higher IPN epimerase activity than the untransformed A. chrysogenum C10, and produced 80 to 100% more cephalosporin C and deacetylcephalosporin C than the parental strain. A second class of transformants, including TMCD2, TMCD32 and TMCD39, in contrast, showed a drastic reduction in cephalosporin biosynthesis relative to the untransformed control. These transformants had no detectable IPN epimerase activity and did not produce cephalosporin C or deacetylcephalosporin C. They also expressed both endogenous and exogenous cefD2 genes only after long periods (72–96 h) of incubation, as shown by Northern analysis, and were impaired in mycelial branching in liquid cultures. The negative effect of amplification of the cefD1 - cefD2 gene cluster in this second class of transformants is not correlated with high gene dosage, but appears to be due to exogenous DNA integration into a specific locus, which results in a pleiotropic effect on growth and cefD2 expression. Communicated by P. J. Punt     

Combining co-culturing of Paenibacillus strains and Vitreoscilla hemoglobin expression as a strategy to improve biodesulfurization

Enhancement of the desulfurization activities of Paenibacillus strains 32O-W and 32O-Y were investigated using dibenzothiophene (DBT) and DBT sulfone (DBTS) as sources of sulphur in growth experiments. Strains 32O-W, 32O-Y and their co-culture (32O-W plus 32O-Y), and Vitreoscilla hemoglobin (VHb) expressing recombinant strain 32O-Yvgb and its co-culture with strain 32O-W were grown at varying concentrations (0·1–2 mmol l⁻¹) of DBT or DBTS for 96 h, and desulfurization measured by production of 2-hydroxybiphenyl (2-HBP) and disappearance of DBT or DBTS. Of the four cultures grown with DBT as sulphur source, the best growth occurred for the 32O-Yvgb plus 32O-W co-culture at 0·1 and 0·5 mmol l⁻¹ DBT. Although the presence of vgb provided no consistent advantage regarding growth on DBTS, strain 32O-W, as predicted by previous work, was shown to contain a partial 4S desulfurization pathway allowing it to metabolize this 4S pathway intermediate.     

Sequence analysis and gene amplification study of the penicillin biosynthesis gene cluster from different strains of <Emphasis Type="Italic">Penicillium chrysogenum</Emphasis>

Industrial strains of Penicillium chrysogenum possess many genomic changes leading to higher levels of penicillin. In this work several production and wild-type strains of Penicillium chrysogenum were used in comparative nucleotide sequence analysis of the biosynthesis cluster. The alignments confirmed sequence conservation not only in promoter regions of the biosynthesis genes but also throughout the entire 44.7-kbp genomic fragment comprising the whole biosynthesis cluster with 15.5-kbp and 13.1-kbp flanking regions. As another titre-enhancing mechanism we subsequently examined gene dosage in two production strains introduced here, NMU2/40 and B14. Quantitative real-time PCR and Southern blot analysis showed the amplification of the biosynthesis genes in both these strains. Through the real-time PCR method the exact copy number was estimated for each of the pcbAB, pcbC and penDE genes. The equal pool of all three genes per genome was confirmed for the both production strains indicating that in these strains the entire penicillin cluster has been amplified as an intact element. Penicillium chrysogenum NMU2/40 was found to carry four copies of the cluster, while six copies were estimated for B14. This also proves the contribution of the additional titre-enhancing mechanisms in both strains, since the industrial data referred much higher production of these strains compared with the single copy reference strain NRRL 1951.     

Impact of the Penicillium chrysogenum genome on industrial production of metabolites

The genome sequence of Penicillium chrysogenum has initiated a range of fundamental studies, deciphering the genetic secrets of the industrial penicillin producer. More than 60 years of classical strain improvement has resulted in major but delicate rebalancing of the intracellular metabolism leading to the impressive penicillin titres of the current production strains. Several leads for further improvement are being followed up, including the use of P. chrysogenum as a cell factory for other products than β-lactam antibiotics.     

Functional analysis of MFS protein CefT involved in the transport of beta-lactam antibiotics in Acremonium chrysogenum and Saccharomyces cerevisiae

Vectors for the expression of MFS transporter CefT of Acremonium chrysogenum—a producer of beta-lactam antibiotic cephalosporin C—and in Saccharomyces cerevisiae as a fusion with the cyan fluorescent protein (CFP) have been generated. The subcellular localization of the CefT-CFP hybrid protein in yeast cells has been investigated. It was shown that the CefT-CFP hybrid protein is capable of complementation of the qdr3, tpo1, and tpo3 genes encoding for orthologous MFS transporters of Saccharomycetes, making the corresponding strains resistant to spermidine, ethidium bromide, and hygromycin B. High-producing strain A. chrysogenum VKM F 4081D, expressing the cefT-cfp fusion construct, was obtained by an agrobacteria mediated transformation. It was also shown that the constitutive expression of cefT in A. chrysogenum VKM F-4081D led to a change in the biosynthetic profiles of cephalosporin C and its precursors. This resulted in a 25–35% decrease in the amount of the final product accumulated in the cultural liquid with a simultaneous increase in the concentration of its intermediates.     

Regulation and compartmentalization of β‐lactam biosynthesis

Penicillins and cephalosporins are β-lactam antibiotics widely used in human medicine. The biosynthesis of these compounds starts by the condensation of the amino acids l -α-aminoadipic acid, l -cysteine and l -valine to form the tripeptide δ-l -α-aminoadipyl-l -cysteinyl-d -valine catalysed by the non-ribosomal peptide ‘ACV synthetase’. Subsequently, this tripeptide is cyclized to isopenicillin N that in Penicillium is converted to hydrophobic penicillins, e.g. benzylpenicillin. In Acremonium and in streptomycetes, isopenicillin N is later isomerized to penicillin N and finally converted to cephalosporin. Expression of genes of the penicillin (pcbAB, pcbC, pendDE) and cephalosporin clusters (pcbAB, pcbC, cefD1, cefD2, cefEF, cefG) is controlled by pleitropic regulators including LaeA, a methylase involved in heterochromatin rearrangement. The enzymes catalysing the last two steps of penicillin biosynthesis (phenylacetyl-CoA ligase and isopenicillin N acyltransferase) are located in microbodies, as shown by immunoelectron microscopy and microbodies proteome analyses. Similarly, the Acremonium two-component CefD1–CefD2 epimerization system is also located in microbodies. This compartmentalization implies intracellular transport of isopenicillin N (in the penicillin pathway) or isopenicillin N and penicillin N in the cephalosporin route. Two transporters of the MFS family cefT and cefM are involved in transport of intermediates and/or secretion of cephalosporins. However, there is no known transporter of benzylpenicillin despite its large production in industrial strains.     

Resorcinol degradation by a <Emphasis Type="Italic">Penicillium chrysogenum</Emphasis> strain under osmotic stress: mono and binary substrate matrices with phenol

A phenol-degrading Penicillium chrysogenum strain previously isolated from a salt mine was able to grow at 1,000 mg l⁻¹ of resorcinol on solid medium. The aerobic degradation of resorcinol by P. chrysogenum CLONA2 was studied in batch cultures in minimal mineral medium with 58.5 g l⁻¹ of sodium chloride using resorcinol as the sole carbon source. The fungal strain showed the ability to degrade up to 250 mg l⁻¹ of resorcinol. Resorcinol and phenol efficiency degradation by P. chrysogenum CLONA2 was compared. This strain removes phenol faster than resorcinol. When phenol and resorcinol were in binary substrate matrices, phenol enhanced resorcinol degradation, and organic load decreased with respect to the mono substrate matrices. The acute toxicity of phenol and resorcinol, individually and in combination, to Artemia franciscana larvae has been verified before and after the bioremediation process with P. chrysogenum CLONA2. The remediation process was effective in mono and binary substrate systems.     

Metabolic engineering of Aeromonas hydrophila for the enhanced production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)

Wild-type Aeromonas hydrophila 4AK4 produced 35–45 wt.% poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) consisting of 10–15 mol% 3-hydroxyhexanoate (3HHx). To enhance PHBHHx production, vgb gene encoding Vitreoscilla haemoglobin or fadD gene encoding Escherichia coli acyl-CoA synthase was co-expressed with polyhydroxyalkanoates (PHA) synthesis-related genes including phbAB from Wautersia eutropha and phaPCJ from A. hydrophila. Expression of vgb increased PHBHHx content from 46 to 53 wt.% without affecting the polymer monomers composition, whereas fadD increased both PHBHHx content from 46 to 64 wt.% and its 3HHx fraction from 15 to 24 mol%. Co-expression of vgb or fadD gene with PHA-synthesis-related genes generally increased PHBHHx content over 60 wt.%. Co-expression of phbAB with vgb increased PHBHHx content and concentration up to about 70 wt.% and 4.0 g l⁻¹, respectively. Fermentor study also showed that in the recombinants harboring vgb, CDW, PHBHHx concentration and productivity were significantly elevated up to 54 g l⁻¹, 28.5 g l⁻¹ and 0.791 g l⁻¹ h⁻¹, respectively, suggesting that vgb could promote PHA synthesis. In this strain, lac promoter could be used to constitutively express foreign genes such as phbA and phbB encoding β-ketothiolase and NADPH-dependent acetoacetyl-CoA reductase of W. eutropha, respectively, without use of IPTG. The results showed that combined expression of different genes was a successful strategy to enhance PHA production, which could be useful for strain development to construct other recombinant PHA-producing strains.     

Effects of Carbon Source and Vitreoscilla Hemoglobin (VHb) on the Production of β-Galactosidase in Enterobacter aerogenes

At fixed concentration (0.5%), lactose and galactose acted as inducers while glucose and other tested carbon sugars showed repression effects on β-galactosidase production in Enterobacter aerogenes strain. The expression of Vitreoscilla hemoglobin gene (vgb) in this bacterial strain managed to overcome the repression effects as well as improving the induction of β-galactosidase formation by carbon sources. In parallel, the bacterial O₂ consumption was increased correspondingly to the vgb induction of β-galactosidase synthesis. When Enterobacter aerogenes strains were grown at the incubation temperature 42°C, about 5-fold higher enzyme productivity was obtained than with a similar incubation at 37°C. The bacterial growth expressed as biomass yield had a different optimum temperature and was not influenced to the same extent by variations in the carbon sources. These data are discussed in terms of proposed enhancement in β-galactosidase productivity by vgb expression as well as its significance to improve the technology of whey processing.     

Expression of <Emphasis Type="Italic">Vitreoscilla</Emphasis> hemoglobin in <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> improve the cell density and insecticidal crystal proteins yield

The Vitreoscilla hemoglobin (VHb) gene (vgb) was integrated into the chromosome of Bacillus thuringiensis BMB171 using integrative vector pEG491. The production of VHb was confirmed by CO-difference spectra analysis. Fermentation experiments results showed that with the production of VHb, the critical oxygen concentration (COC) of the host strain was reduced from 18 to 12%. The maximum viable cell counts of the VHb⁺ strain in high, middle, and low aeration/agitation fermentations were 0.94-, 1.23-, and 1.59-fold of those of the VHb⁻ strain, respectively. Under the same conditions, the yields of insecticidal crystal proteins (ICP) by VHb⁺ strain were 1.22-, 1.63-, and 3.13-fold of those of the VHb⁻ strain. The production of VHb also accelerated the formation of ICP and spores. These results indicated that the production of VHb could improve the cell density and ICP yield of B. thuringiensis, especially under low aeration/agitation condition.