Shuai Zhao

Modern sugarcane cultivars are allo‐autopolyploids and aneuploids, which pose great challenges for research and breeding to meet global demand. Recent breakthroughs in gene‐editing technologies offer the potential to expedite the development of high‐yield stress‐resilient sugarcane, a vegetatively propagated crop. However, the limited valuable targets, coupled with complex transgenic methodologies for verifying gene function, hamper this progress. Protoplasts enable rapid gene introduction and function validation, yet an efficient protoplast expression system for elite sugarcane cultivars is currently lacking. Here, we developed a protoplast isolation and transfection method using tissue‐cultured GT42, a primary sugarcane cultivar in China, yielding 1.41 × 10 ⁷ protoplasts/g fresh weight with 78.74% viability and 39.01% transfection efficiency. This system proved effective for various assays, including GCaMP6‐based calcium imaging. Using this system, we showed that abscisic acid biosynthesis gene ScNCED3 acts as an early drought signal, positively regulated by ScPYL4‐12, ScOST1‐12 and ScMPK6‐11, whereas salicylic acid (SA) enhances ScPR1 expression. Notably, the ScNPRs exhibited opposite functionalities compared to those in Arabidopsis , with ScNPR1 negatively and ScNPR3/4 positively activating ScPR1 expression upon SA treatment. Together, we established an efficient protoplast expression system using GT42 sugarcane culm, providing a valuable tool for studying sugarcane gene function under stress.

Cellulase plays an irreplaceable role in biomanufacturing using plant biomass as feedstock. However, improving cellulase production by fungi through manipulation of upstream open reading frames (uORFs) in the 5′-untranslated regions (5′-UTR) of cellulase genes has been less frequently explored. This study aimed to screen uORFs in the 5′-UTR of cellulase genes in Penicillium oxalicum, identify functional uORFs in the 5′-UTR of the eg1 gene which encodes a key endo-β-1,4-glucanase (EG) in P. oxalicum, and enhance fungal cellulase production through uORF modifications. Among the 25 cellulase genes examined in P. oxalicum strain HP7-1, 23 contained uORFs in their 5′-UTR. Seven uORFs were annotated in the 5′-UTR of the eg1 gene. A uORF-green fluorescent protein (GFP) reporter system demonstrated that uORF1 and uORF3 inhibited, while uORF7 enhances, GFP abundance. Overexpression of eg1 containing uORF1 or uORF3 variants where the start codon of the uORF was mutated to AAG in P. oxalicum led to a significant 91.7 % and 62.1 % average increase in carboxymethyl cellulase production after 4 days of induction compared to the start strain ΔPoxKu70. Real-time quantitative reverse transcription-polymerase chain reaction, mRNA stability determination, and in vitro translation experiments collectively revealed that these three uORFs influence the mRNA stability of the downstream mORF, but not translation efficiency. These findings highlight the critical role of uORFs in regulating gene expression during fungal enzyme biosynthesis and offer a valuable alternative strategy for improving enzyme production.

Starch is an abundant, low-cost, renewable carbohydrate, yet conventional enzymatic biorefining requires energy-intensive liquefaction and saccharification that reduce economic viability. Raw starch-degrading enzymes (RSDEs) promise low-temperature, direct granular hydrolysis, but native forms show low catalytic efficiency, poor stability, and limited yields. This review summarizes recent advances in the discovery, expression, modification, and application of RSDEs. It discusses strategies to enhance RSDE expression through genetic manipulation of regulatory factors, particularly transcription factors governing amylolytic gene networks. Subsequently, it details molecular modification approaches, including protein engineering and genetic modification, to improve RSDE performance and production. Furthermore, we highlight the development of engineered microbial strains with high-yield of RSDE for scalable RSDE production and application. Finally, the future development and prospects of next-generation RSDEs are proposed. This review provides a comprehensive foundation for industrial implementation of RSDEs and offers strategic insights into the development of high-performance enzymes and robust production platforms.

Nitriles exhibit acute cytotoxicity to human and animal cells. Nitrilase is a green biocatalyst that can directly convert nitrile into nontoxic carboxylic acids and ammonia. However, the nitrilases capable of degrading 3-butenenitrile and 4-pentenenitrile derived from glucosinolate present in rapeseed meals are still limited. This study expressed the nitrilase 2 gene from bacterium Paraburkholderia graminis (PgNIT2) in Escherichia coli. The purified recombinant nitrilase rPgNIT2 showed specific activities of 5.94 U/mg and 6.66 U/mg when using 3-butenenitrile and 4-pentenenitrile as substrates at the optimal pH 7.0 and temperature 45 °C. The substrate binding pocket of PgNIT2 comprising 12 amino acids was required for rPgNIT2 activity. Remarkably, the specific activity of mutant enzyme A190I increased by 68.3% and 57.3%, respectively, against 3-butenenitrile and 4-pentenenitrile compared with the wild-type enzyme. Furthermore, the A190I showed improved thermostability and hydrolysis ability towards 3-butenenitrile and 4-pentenenitrile compared with rPgNIT2 but shared the same optimal pH and temperature. These results provided theoretical guidance for engineering of bacterial nitrilases used for rapeseed meal detoxification.

Rapeseed meal is severely restricted in its utilization as unconventional animal feed due to anti-nutritive compounds, such as glucosinolate, that are degraded to toxic nitriles such as 3-butenenitrile and 4-pentenenitrile in animals. Few studies on nitrilases that can degrade glucosinolate-derived nitriles have been reported thus far. In the present study, a nitrilase gene GiNIT from Gibberella intermedia was over-expressed in Escherichia coli and the purified recombinant nitrilase rGiNIT showed specific activities of 134.48 U/mg and 122.16 U/mg when using 3-butenenitrile and 4-pentenenitrile as substrates at the optimal pH, 7.5, and temperature, 45 °C, which is the highest reported in the literature. The conversion of 3-butenenitrile and 4-pentenenitrile by rGiNIT reached 81.89% and 80.23% after hydrolysis for 15 min and 300 min, respectively. Site-directed mutagenesis and molecular docking analysis revealed that the catalytic ability of rGiNIT depended on the substrate binding pocket comprising 13 key amino acid residues. These results provide a potential enzyme resource for rapeseed meal detoxification and theoretical guidance for protein engineering.

Filamentous fungi can produce raw-starch-degrading enzyme, however, regulation of production of raw-starch-degrading enzyme remains poorly understood thus far. Here, two novel transcription factors raw-starch-degrading enzyme regulator D (RsrD) and raw-starch-degrading enzyme regulator E (RsrE) were identified to participate in the production of raw-starch-degrading enzyme in Penicillium oxalicum . Individual knockout of rsrD and rsrE in the parental strain Δ ku70 resulted in 31.1%–92.9% reduced activity of raw-starch-degrading enzyme when cultivated in the presence of commercial starch from corn. RsrD and RsrE contained a basic leucine zipper and a Zn2Cys6-type DNA-binding domain, respectively, but with unknown functions. RsrD and RsrE dynamically regulated the expression of genes encoding major amylases over time, including raw-starch-degrading glucoamylase gene PoxGA15A and α-amylase gene amy13A . Interestingly, RsrD and RsrE regulated each other at transcriptional level, through binding to their own promoter regions; nevertheless, both failed to bind to the promoter regions of PoxGA15A and amy13A , as well as the known regulatory genes for regulation of amylase gene expression. RsrD appears to play an epistatic role in the module RsrD-RsrE on regulation of amylase gene expression. This study reveals a novel regulatory pathway of fungal production of raw-starch-degrading enzyme. IMPORTANCE: To survive via combating with complex extracellular environment, filamentous fungi can secrete plant polysaccharide-degrading enzymes that can efficiently hydrolyze plant polysaccharide into glucose or other mono- and disaccharides, for their nutrients. Among the plant polysaccharide-degrading enzymes, raw-starch-degrading enzymes directly degrade and convert hetero-polymeric starch into glucose and oligosaccharides below starch gelatinization temperature, which can be applied in industrial biorefinery to save cost. However, the regulatory mechanism of production of raw-starch-degrading enzyme in fungi remains unknown thus far. Here, we showed that two novel transcription factors raw-starch-degrading enzyme regulator D (RsrD) and raw-starch-degrading enzyme regulator E (RsrE) positively regulate the production of raw-starch-degrading enzyme by Penicillium oxalicum . RsrD and RsrE indirectly control the expression of genes encoding enzymes with amylase activity but directly regulate each other at transcriptional level. These findings expand diversity of gene expression regulation in fungi.

Filamentous fungi produce polysaccharide-degrading enzymes, which is controlled by poorly understood transcriptional circuits. Here we show that a circuit comprising RsrC-RsrA-RsrB (Rsr: production of raw-starch-degrading enzyme regulator) that positively regulates production of raw starch-degrading enzymes in Penicillium oxalicum. Transcription factor (TF) RsrA is essential for biosynthesis of raw starch-degrading enzymes. RsrB and RsrC containing Zn2Cys6- and C2H2-zinc finger domains, act downstream and upstream of RsrA, respectively. RsrA activates rsrB transcription, and three nucleotides (G^-286, G^-287 and G^-292) of rsrB promoter region are required for RsrA, in terms of TF, for binding. RsrB_165−271 binds to DNA sequence 5’-TCGATCAGGCACGCC-3’ in the promoter region of the gene encoding key raw-starch-degrading enzyme PoxGA15A. RsrC specifically binds rsrA promoter, but not amylase genes, to positively regulate the expression of rsrA and the production of raw starch-degrading enzymes. These findings expand complex regulatory network of fungal raw starch-degrading enzyme biosynthesis.

Filamentous fungi produce polysaccharide-degrading enzymes governing tolerance to changing environments, and this is controlled by a poorly understood transcriptional circuit. Here, genome-wide screening and genetic analyses reveal a novel regulatory circuit comprising RsrC-RsrA-RsrB that positively regulates the production of raw starch-degrading enzymes (RSDEs) in Penicillium oxalicum. Transcription factor (TF) RsrA is essential for biosynthesis of RSDEs. Two novel TFs, RsrB and RsrC, containing Zn2Cys6 and C2H2 zinc finger domains, respectively, act downstream and upstream of RsrA. RsrA activates transcription of rsrB, and three nucleotides (G^-286, G^-287 and G^-292) are required for RsrA binding to rsrB. Polypeptide RsrB_165-271 binds the conserved DNA sequence 5’-KBKWYSNRKNDVVBS-3’ in the promoters of genes encoding major amylases. RsrC specifically binds rsrA promoter via bases -850 to -825, but not the promoters of amylase genes. This regulatory circuit influences mycelial growth and conidia production. The findings expand the complex regulatory network of fungal RSDE biosynthesis and development.