Filamentous fungi play important roles in the production of plant cell-wall degrading enzymes. In recent years, homologous recombinant technologies have contributed significantly to improved enzymes production and system design of genetically manipulated strains. When introducing multiple gene deletions, we need a robust and convenient way to control selectable marker genes, especially when only a limited number of markers are available in filamentous fungi. Integration after transformation is predominantly nonhomologous in most fungi other than yeast. Fungal strains deficient in the non-homologous end-joining (NHEJ) pathway have limitations associated with gene function analyses despite they are excellent recipient strains for gene targets. We describe strategies and methods to address these challenges above and leverage the power of resilient NHEJ deficiency strains. We have established a foolproof light-inducible platform for one-step unmarked genetic modification in industrial eukaryotic microorganisms designated as ‘LML 3.0’, and an on-off control protocol of NHEJ pathway called ‘OFN 1.0’, using a synthetic light-switchable transactivation to control Cre recombinase-based excision and inversion. The methods provide a one-step strategy to sequentially modify genes without introducing selectable markers and NHEJ-deficiency. The strategies can be used to manipulate many biological processes in a wide range of eukaryotic cells.

Background: The filamentous fungus Trichoderma reesei is a major workhorse employed to produce cellulase, which hydrolyzes lignocellulosic biomass for the production of cellulosic ethanol and bio-based products. However, the economic efficiency of biorefineries is still low. Results: In this study, the truncation of cellulase activator ACE3 was identified and characterized in T. reesei classical mutant NG14 and its direct descendants for the first time. We demonstrated that the truncated ACE3 is the crucial cause of cellulase hyper-production in T. reesei NG14 branch. Replacing the native ACE3 with truncated ACE3 in other T. reesei strains remarkably improves cellulase production. By truncating ACE3, we engineered a T. reesei mutant, PC-3-7-A723, capable of producing more cellulase than other strains. In a 30-L fermenter, fed-batch fermentation with PC-3-7-A723 drastically increased the maximum cellulase titer (FPase) to 102.63 IU/mL at 240 h, which constitutes a 20-30% improvement to that of the parental strain PC-3-7. Conclusions: This work characterized the function of truncated ACE3 and demonstrated that analysis of classical mutants allows rational engineering of mutant strains with improved cellulase production necessary to process lignocellulosic biomass. Our rational engineering strategy might be useful for enhancing the production of other bio-based products.

Acetoin (AC) is a volatile platform compound with various potential industrial applications. AC contains two stereoisomeric forms: (3)-AC and (3)-AC. Optically pure AC is an important potential intermediate and widely used as a precursor to synthesize novel optically active materials. In this study, chiral (3)-AC production from -2,3-butanediol (-2,3-BD) was obtained using recombinant cells co-expressing-2,3-butanediol dehydrogenase (-2,3-BDH), NADH oxidase (NOX), and hemoglobin protein (VHB) from sp. T241,, and , respectively. The new biocatalyst of/pET--- was developed and the bioconversion conditions were optimized. Under the optimal conditions, 86.74 g/l of (3)-AC with the productivity of 3.61 g/l/h and the stereoisomeric purity of 97.89% was achieved from 93.73 g/l -2,3-BD using the whole-cell biocatalyst. The yield and productivity were new records for (3)-AC production. The results exhibit the industrial potential for (3)-AC production via whole-cell biocatalysis.

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