Trichoderma reesei possesses a robust post-translational modification system and protein secretion capacity, making it one primary host producing enzymes such as cellulase.To enhance its gene-editing efficiency, the previously constructed recombinant T.reesei CJ2095 expressing recombinant human lactoferrin was used as an initial strain in this study.The key gene for non-homologous end joining, the DNA repair protein gene ku80, was knocked out, resulting in the recombinant strain T.reesei CJ2095 Δku80 with significantly improved gene-editing efficiency.The gene-editing efficiency was increased from 1.04% of the control strain to 7.14%.To enable the recycling of the orotidine-5′-phosphate decarboxylase gene pyr4 as a selection marker, the pyr4 gene was knocked out from the genome of T.reesei CJ2095 Δku80, and the recombinant strain T.reesei CJ2095 Δku80Δpyr4 was constructed.To further improve homologous recombination efficiency, the endogenous homologous recombination genes rad51 or rad52 in T.reesei were over-expressed.Overexpression of the rad52 gene resulted in a recombinant strain T.reesei CJ2095 Δku80Δpyr4::rad52 with a homologous recombination efficiency of 43.75%, which was 42-fold higher than that of the initial strain.The high gene-editing efficiency of T.reesei CJ2095 Δku80Δpyr4::rad52 was further verified by overexpressing the xylanase regulator 1 gene xyr1 in the genome, which significantly increased the yield of recombinant human lactoferrin.The yield of recombinant human lactoferrin in T.reesei CJ2095 Δku80Δpyr4::rad52::xyr1 was increased by 22.21%, reaching 234.64 mg/L.This is the first report that a gene-editing strategy has been used to enhance the production level of recombinant human lactoferrin in T.reesei.
HU Chuanjun
,
YANG Haiquan
,
XIA Yuanyuan
,
SHEN Wei
,
CHEN Xianzhong
. Engineering Trichoderma reesei to enhance its gene-editing efficiency[J]. Food and Fermentation Industries, 2026
, 52(3)
: 27
-36
.
DOI: 10.13995/j.cnki.11-1802/ts.042949
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