Acthi, a thiazole biosynthesis enzyme, is essential for thiamine biosynthesis and CPC production in Acremonium chrysogenum

Abstract

BackgroundThe filamentous fungus Acremonium chrysogenum is an important industrial fungus and is used in the production of the β-lactam antibiotic cephalosporin C. Little is known regarding the molecular and biological mechanisms of how this industrial strain was improved by mutagenesis and molecular breeding. Comparative proteomics is one of the most powerful methods to evaluate the influence of gene expression on metabolite production.ResultsIn this study, we used comparative proteomics to investigate the molecular mechanisms involved in the biosynthesis of cephalosporin C between a high-producer (HY) strain and a wide-type (WT) strain. We found that the expression levels of thiamine biosynthesis-related enzymes, including the thiazole biosynthesis enzyme (Acthi), pyruvate oxidase, flavin adenine dinucleotide (FAD)-dependent oxidoreductase and sulfur carrier protein-thiS, were up-regulated in the HY strain. An Acthi-silencing mutant of the WT strain grew poorly on chemically defined medium (MMC) in the absence of thiamine, and its growth was recovered on MMC medium supplemented with thiamine. The intracellular thiamine content was changed in the Acthi silencing or over-expression mutants. In addition, we demonstrated that the manipulation of the Acthi gene can affect the hyphal growth of Acremonium chrysogenum, the transcription levels of cephalosporin C biosynthetic genes, the quantification levels of precursor amino acids for cephalosporin C synthesis and the expression levels of thiamine diphosphate-dependent enzymes. Over-expression of Acthi can significantly increase the cephalosporin C yield in both the WT strain and the HY mutant strain.ConclusionsUsing comparative proteomics, four differently expressed proteins were exploited, whose functions may be involved in thiamine diphosphate metabolism. Among these proteins, the thiazole biosynthesis enzyme (ActhiS) may play an important role in cephalosporin C biosynthesis. Our studies suggested that Acthi might be involved in the transcriptional regulation of cephalosporin C biosynthesis. Therefore, the thiamine metabolic pathway could be a potential target for the molecular breeding of this cephalosporin C producer for industrial applications.

Inducible promoters and functional genomic approaches for the genetic engineering of filamentous fungi

“…They showed an overrepresentation of enzymes involved in penicillin precursor and energy metabolism, while proteins involved in stress response, virulence, and the biosynthesis of other secondary metabolites were nearly lost. A similar comparison with A. chrysogenum strains also identified comparable changes (Liu et al 2015 ). Since thiamine biosynthesis enzymes are overrepresented in the production strain, this metabolism seems to be beneficial for cephalosporin C biosynthesis in this fungus.…”

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

“…Whenever possible, these defects may be identified and corrected using modern “omics” techniques, systems biology and synthetic biology approaches, metabolic modeling, genome editing, reverse genetics etc [ 68 , 69 ]. Successful examples of the application of this strategy towards metabolic engineering of industrial beta-lactam producing strains include overexpression of cefG gene [ 62 ], introduction of a truncated gene copy for PacC transcription factor, modulation of strain morphology through manipulation with Acatg1 [ 9 ] and Acthi1 [ 10 ] genes, enhancing oxygen uptake by expression of bacterial hemoglobin gene [ 19 ].…”

“…A number of different factors and processes which can also significantly affect CPC biosynthesis in A . chrysogenum were investigated, such as reactive oxygen species [ 6 ], autophagy [ 7 – 9 ], sulfur biosynthesis and endogenous S-adenosylmethionine content [ 10 , 11 ], transcription factors [ 12 – 15 ], etc. These achievements, together with the development of methods for genetic manipulation and “omics” technologies applied to A .…”

“…The "late cluster" (chromosome I) encodes two genes for the final steps of CPC biosynthesis, cefEF (encodes deacetoxycephalosporin C synthetase/ hydroxylase) and cefG (encodes deacetylcephalosporin-C acetyltransferase), responsible for three enzymatic activities with the sequential conversion of PenN to deacetoxycephalosporin C (DAOC), then deacetylcephalosporin C (DAC) and finally to CPC [4,5]. A number of different factors and processes which can also significantly affect CPC biosynthesis in A. chrysogenum were investigated, such as reactive oxygen species [6], autophagy [7][8][9], sulfur biosynthesis and endogenous S-adenosylmethionine content [10,11], transcription factors [12][13][14][15], etc. These achievements, together with the development of methods for genetic manipulation and "omics" technologies applied to A. chrysogenum [16,17], opened new opportunities for improvement of industrial strains [18,19].…”

Study on genetic engineering of Acremonium chrysogenum , the cephalosporin C producer

“…Our lab cloned a putative thiazole biosynthesis gene Ac thi and found knock out of Ac thi in A. chrysogenum reduced the intracellular concentration of thiamine and CPC biosynthesis precursor amino acids, while the formation of conidia was also decreased. Overexpression of Ac thi can restore the reduction, upregulate the transcriptions of CPC biosynthetic genes and increase the production of CPC [47] .…”