Constitutive hyperproduction of sorbicillinoids in Trichoderma reesei ZC121

Chengcheng Li,Wei Sun,Fu-Gen Wu,Zhihua Zhou,Shaoxun Yuan,Fengming Lin,Zhan Chen

doi:10.1186/s13068-018-1296-4

Abstract

BackgroundIn addition to its outstanding cellulase production ability, Trichoderma reesei produces a wide variety of valuable secondary metabolites, the production of which has not received much attention to date. Among them, sorbicillinoids, a large group of hexaketide secondary metabolites derived from polyketides, are drawing a growing interest from researchers because they exhibit a variety of important biological functions, including anticancer, antioxidant, antiviral, and antimicrobial properties. The development of fungi strains with constitutive, hyperproduction of sorbicillinoids is thus desired for future industry application but is not well-studied. Moreover, although T. reesei has been demonstrated to produce sorbicillinoids with the corresponding gene cluster and biosynthesis pathway proposed, the underlying molecular mechanism governing sorbicillinoid biosynthesis remains unknown.ResultsRecombinant T. reesei ZC121 was constructed from strain RUT-C30 by the insertion of the gene 12121-knockout cassette at the telomere of T. reesei chromosome IV in consideration of the off-target mutagenesis encountered during the unsuccessful deletion of gene 121121. Strain ZC121, when grown on cellulose, showed a sharp reduction of cellulase production, but yet a remarkable enhancement of sorbicillinoids production as compared to strain RUT-C30. The hyperproduction of sorbicillinoids is a constitutive process, independent of culture conditions such as carbon source, light, pH, and temperature. To the best of our knowledge, strain ZC121 displays record sorbicillinoid production levels when grown on both glucose and cellulose. Sorbicillinol and bisvertinolone are the two major sorbicillinoid compounds produced. ZC121 displayed a different morphology and markedly reduced sporulation compared to RUT-C30 but had a similar growth rate and biomass. Transcriptome analysis showed that most genes involved in cellulase production were downregulated significantly in ZC121 grown on cellulose, whereas remarkably all genes in the sorbicillinoid gene cluster were upregulated on both cellulose and glucose.ConclusionA constitutive sorbicillinoid-hyperproduction strain T. reesei ZC121 was obtained by off-target mutagenesis, displaying an overwhelming shift from cellulase production to sorbicillinoid production on cellulose, leading to a record for sorbicillinoid production. For the first time, T. reesei degraded cellulose to produce platform chemical compounds other than protein in high yield. We propose that the off-target mutagenesis occurring at the telomere region might cause chromosome remodeling and subsequently alter the cell structure and the global gene expression pattern of strain ZC121, as shown by phenotype profiling and comparative transcriptome analysis of ZC121. Overall, T. reesei ZC121 holds great promise for the industrial production of sorbicillinoids and serves as a good model to explore the regulation mechanism of sorbicillinoids’ biosynthesis.

Highlights

In addition to its outstanding cellulase production ability, Trichoderma reesei produces a wide variety of valuable secondary metabolites, the production of which has not received much attention to date
T. reesei RUT-C30 and its derivatives were cultured on potato dextrose agar (PDA) plates at 28 °C with mixing at 200 rpm for conidia production and in Trichoderma minimal media (TMM) [29] with 2% (w/t) cellulose or other carbon sources for cellulase and sorbicillinoid production
We attempted to knockout gene 121121 in T. reesei RUT-C30 using homologous recombination mediated by Agrobacterium tumefaciens-mediated transformation (AMT) (Fig. 1a and Additional file 1: Fig. S1)

Summary

Introduction

In addition to its outstanding cellulase production ability, Trichoderma reesei produces a wide variety of valuable secondary metabolites, the production of which has not received much attention to date. Sorbicillinoids are produced and secreted by both marine and terrestrial ascomycetes, including Trichoderma [9], Aspergillus [10], Penicillium [11], Streptomyces [1], Acremonium [12], Paecilomyces [13], and Eurotiomycete [14] Most of these compounds possess the characteristic C1–C6 sorbyl sidechain and bi- or tri-cyclic frameworks that are extremely complex and highly oxygenated. Sorbicillinoids fall into four classes: monomeric sorbicillinoids, bisorbicillinoids, trisorbicillinoids and hybrid sorbicillinoids They have a variety of biological activities, including anticancer [15], antioxidant [16], antiviral [17] and antimicrobial [18], showing promising applications in the agriculture, pharmaceutical, and food industries. Most work was aimed at eliminating yellow pigments from fungal fermentation cultures for the production of products like β-lactams [19] and cellulase [9]

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Discussion

Conclusion