The critical role of plasma membrane H+-ATPase activity in cephalosporin C biosynthesis of Acremonium chrysogenum

Abstract

The filamentous fungus Acremonium chrysogenum is the main industrial producer of cephalosporin C (CPC), one of the major precursors for manufacturing of cephalosporin antibiotics. The plasma membrane H + -ATPase (PMA) plays a key role in numerous fungal physiological processes. Previously we observed a decrease of PMA activity in A . chrysogenum overproducing strain RNCM 408D (HY) as compared to the level the wild-type strain A . chrysogenum ATCC 11550. Here we report the relationship between PMA activity and CPC biosynthesis in A . chrysogenum strains. The elevation of PMA activity in HY strain through overexpression of PMA1 from Saccharomyces cerevisiae , under the control of the constitutive gpdA promoter from Aspergillus nidulans , results in a 1.2 to 10-fold decrease in CPC production, shift in beta-lactam intermediates content, and is accompanied by the decrease in cef genes expression in the fermentation process; the characteristic colony morphology on agar media is also changed. The level of PMA activity in A . chrysogenum HY OE :: PMA1 strains has been increased by 50–100%, up to the level observed in WT strain, and was interrelated with ATP consumption; the more PMA activity is elevated, the more ATP level is depleted. The reduced PMA activity in A . chrysogenum HY strain may be one of the selected events during classical strain improvement, aimed at elevating the ATP content available for CPC production.

Polyamines Upregulate Cephalosporin C Production and Expression of β-Lactam Biosynthetic Genes in High-Yielding Acremonium chrysogenum Strain

“…Thus, OD 600 = 0.5 were ~2.7 × 10 7 CFU/mL for the A. chrysogenum WT strain and 1.6 × 10 6 CFU/mL for the A. chrysogenum HY strain. Industrial strains with overproduction of SMs, obtained using classical strain improvement (CSI) programs, often contain unwanted side mutations and "bottlenecks" that negatively affect the fitness of the strain, resistance, productivity, and adaptation to harsh fermentation conditions [2,50,51]. It is possible that the introduction of exogenous PAs could complement or correct some of these negative effects.…”

“…Filamentous fungi (also called molds or moldy fungi) are taxonomically diverse organisms from phylum Ascomycota and Zygomycota with filamentous hyphae and the ability to produce airborne spores or conidia. Improved strains of filamentous fungi are one of the most important sources for producing pharmaceutically significant secondary metabolites (SMs), such as antibiotics, statins, and immunosuppressants [1][2][3][4]. These compounds are one of the most commonly prescribed drugs worldwide [5,6].…”

“…This improved strain typically produces 9-12 g/L of CPC during laboratory fermentation in shake flasks, 200-300 times higher than A. chrysogenum WT. The HY strain is one of the most comprehensively characterized among the improved A. chrysogenum producers of CPC [2,11]. For this strain, the chromosomal rearrangements compared to WT strain were shown; the number of copies and localization of biosynthetic gene clusters (BGCs) of betalactams, the so-called "early" and "late" clusters, were determined [12].…”

“…The A. chrysogenum HY was one of the first highly active producers of SM in filamentous fungi, for which a genetic transformation using Agrobacterium tumefaciens-mediated transformation (ATMT) was developed [14]. Using this technology, we obtained a series of recombinant strains, expressing CefT (MFS beta-lactam transporter from A. chrysogenum) and PMA1 (H + -ATPase the plasma membrane from S. cerevisiae), which made it possible to study in detail the features of transport and energy consumption of beta-lactam biosynthesis in A. chrysogenum [2,15]. The HY strain was also characterized by a shift in several biochemical parameters, particularly the content of five fractions of energy-rich inorganic polyphosphates (polyPs), ATP, and H + -ATPase activity in the plasma membrane [16].…”

High-Yielding Lovastatin Producer Aspergillus terreus Shows Increased Resistance to Inhibitors of Polyamine Biosynthesis

“…As a result of multi-round random mutagenesis and screening, useful mutations are selected, leading to an increase in the yield of the target SM [16]. Along with this, a genetic load accumulates which represents side mutations and is expressed in a general decrease of strains fitness and a reduction in other vital processes [10,17,50]. In response to mutagenic effects, the cell activates its protective resources; PAs are also known to be able to protect cells from free radical damage through direct interaction with reactive oxygen species [51,52].…”