Consolidated bioprocessing (CBP) is a promising strategy for low-cost malic acid production from plant biomass. In this study, we report a comprehensive redox engineering strategy to redirect reducing equivalents towards the reductive tricarboxylic acid (rTCA) pathway for enhanced malic acid production directly from cellulose in the thermophilic fungus Myceliophthora thermophila JG207. Firstly, deletion of key NADH-consuming pathways, including glycerol-3-phosphate dehydrogenase (gpdh) and mitochondrial external NADH dehydrogenases (nde1 and nde2), resulted in a significant increase in the NADH/NAD⁺ ratio and a corresponding 17% increase in malic acid titre. Subsequent removal of alternative oxidase (AOX) further heightened the NADH pool, although it induced oxidative stress and slowed growth. To alleviate redox imbalance, we overexpressed the native transhydrogenase SthA, which effectively converted NADPH to NADH and improved malate production by an additional 6%. Fine-tuning NAD⁺ kinase (UTR1) expression further optimised cofactor homoeostasis, bringing about the highest malate titre of 89.2 g/L, a 38.9% improvement over the starting strain JG207. Finally, overexpression of lytic polysaccharide monooxygenase gene (lpmo) and cellobiose dehydrogenase gene (cdh) enhanced cellulose degradation, shortening the fermentation cycle by two days on Avicel and crushed corncob. This work not only advances the fundamental understanding of redox metabolism in filamentous fungi but also provides a proof-of-concept for sustainable organic acid production from inexpensive lignocellulosic feedstocks.

Various precise gene editing techniques at the DNA/RNA level, driven by clustered regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology, have gained significant prominence. Yet, research on targeted protein editing techniques remains limited. Only a few attempts have been made, including the use of specific proteases and de-O-glycosylating enzymes as editing enzymes. Here, we propose direct editing of N-glycosylated proteins using de-N-glycosylating enzymes to modify N-glycosylation and simultaneously alter the relevant asparagine residue to aspartate in living cells. Selective protein deglycosylation editors were developed by fusing high-affinity protein-targeting peptides with active peptide:N-glycanases (PNGases). Three crucial cell membrane proteins, programmed cell death protein-1 (PD-1), programmed cell death-1 ligand 1 (PD-L1), and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein, were chosen to be tested as a proof of concept. N-linked glycans were removed, and the relevant sites were converted from Asn to Asp in living mammalian cells, destabilizing target proteins and accelerating their degradation. Further investigation focused on SARS-CoV-2 spike protein deglycosylation editing. The collaboration of LCB1-PNGase F (PNGF) effectively reduced syncytia formation, inhibited pseudovirus packaging, and significantly hindered virus entry into host cells, which provides insights for coronavirus disease 2019 (COVID-19) treatment. This tool enables editing protein sequences post-de-N-glycosylation in living human cells, shedding light on protein N-glycosylation functions, and Asn to Asp editing in organisms. It also offers the potential for developing protein degradation technologies.

Protein citrullination involves the deimination of arginine or methylarginine residues in peptide chains to form citrulline by peptidyl arginine deiminases. This process is an important protein post-translational modification that affects molecular structure and function of various proteins, including histones. In recent years, protein citrullination has attracted widespread attention for its influence on gene transcription. Studies on the impact of protein citrullination modification on chromatin structure remodeling and the establishment of gene regulatory networks have made rapid progress. In this review, we briefly summarize the physiological functions of protein citrullination modification. Specifically, we comprehensively outline the latest progress in the study of the role of protein citrullination modification in gene transcription regulation, focusing on the interaction of protein citrullination with other post-translational modifications.