Optimal production of Phanerochaete chrysosporium manganese peroxidases and Trametes sp. C30 laccase hybrid Lac131 in Aspergillus niger for lignin bioconversion

Dai, Ziyu; Robles, Ana L.; Lemmon, Sarah L.; Yuan, Guoliang; Hu, Dehong; Onley, Jenny Rae; Jia, Jiayuan; Deng, Kai; Gupta, Kshitiz; Northen, Trent R.; Simmons, Blake A.; Baker, Scott E.; Magnuson, Jon K.; Kim, Joonhoon

doi:10.1186/s13068-025-02690-x

Title: Optimal production of Phanerochaete chrysosporium manganese peroxidases and Trametes sp. C30 laccase hybrid Lac131 in Aspergillus niger for lignin bioconversion

Journal Article · Mon Oct 06 00:00:00 UTC 2025 · Biotechnology for Biofuels and Bioproducts

DOI: https://doi.org/10.1186/s13068-025-02690-x · OSTI ID:2999146

Dai, Ziyu ^[1]; Robles, Ana L. ^[2]; Lemmon, Sarah L. ^[2]; Yuan, Guoliang ^[2]; Hu, Dehong ^[2]; Onley, Jenny Rae ^[3]; Jia, Jiayuan ^[1];

^[3]; Gupta, Kshitiz ^[4]; Northen, Trent R. ^[4]; Simmons, Blake A. ^[5]; Baker, Scott E. ^[1]; Magnuson, Jon K. ^[1]; Kim, Joonhoon ^[1]

Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)
Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)
Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Sandia National Laboratories (SNL-CA), Livermore, CA (United States)
Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Livermore National Laboratory (LLNL), Livermore, CA (United States)
Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Background Incorporating the production of related ligninolytic enzymes into industrial filamentous fungus Aspergillus niger will enhance the bioconversion of lignocelluloses to various chemical products. Results In this study, transgenic expression of Phanerochaete chrysosporium manganese peroxidases (mnps) and Trametes sp. C30 laccase hybrid Lac131 (lac131) were examined and optimized in A. niger 11414 prtT∆ strain. Five mnps (mnp1, mnp2, mnp3, mnp4, and mnp5) and lac131 genes were expressed separately or in combination. The transgenic strain containing the entire mnp2 genomic coding sequence (gmnp2) exhibited the highest mnP activity among the five mnp over-expression strains in the modified minimal medium (mMM) with addition of 5 g/L bovine hemoglobin (bHg). We examined the effects of hemin and bHg on mnP production in the gmnp2 strain cultures and found that at least 1 g/L bHg was required, while hemin was not. Culture conditions for mnP production were further optimized for the gmnp2 strain and the highest mnP activities were detected in the cultures grown at 25 °C and 200 rpm with an initial pH of 4.5. Effects of soy protein, skim milk, and bovine serum albumin on mnP production were investigated; 5 g/L of soy proteins or skim milk had comparable effects to 2.5 g/L bHg, while cultures with bovine serum albumin had diminished mnP activity. Disruption of both prtT and vsm1 substantially augmented the mnP production and its activity reached 575 U/L. Trametes sp. C30 laccase hybrid lac131 was strongly expressed in either A. niger gmnp2 (1975 U/L) or 11414prtT∆ (3895 U/L) strain. Both mnP and laccase in the culture supernatants effectively decolorized selected phenolic compounds (dyes) and cleaved tagged model lignin dimers. Conclusion The mnP was successfully produced in A. niger by optimizing the culture conditions and host strain. Co-expression of all four mnp genes in the same expression host by multiplex CRISPR will lead to the mnP production reaching levels comparable to P. chrysosporium, while only requiring 36 h at 25 °C. The Lac131 activity in transgenic A. niger strain is 4- to 7-times higher than that in previous studies. Co-production of mnP and laccase in A. niger will enhance the lignin bioconversion efficiency.

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Research Organization:: Sandia National Laboratories (SNL-CA), Livermore, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: AC02-05CH11231; NA0003525

OSTI ID:: 2999146

Report Number(s):: SAND--2025-12846J; 1789459

Journal Information:: Biotechnology for Biofuels and Bioproducts, Journal Name: Biotechnology for Biofuels and Bioproducts Journal Issue: 1 Vol. 18; ISSN 2731-3654

Publisher:: BioMed CentralCopyright Statement

Country of Publication:: United States

Language:: English

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Aspergillus niger
Bovine hemoglobin
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Manganese peroxidase
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Trametes sp. C30
Transcriptional activator of proteases prtT
v-SNARE binding protein VSM

Title: Optimal production of Phanerochaete chrysosporium manganese peroxidases and Trametes sp. C30 laccase hybrid Lac131 in Aspergillus niger for lignin bioconversion

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