γ-Aminobutyric acid (GABA) is a widely distributed non-protein amino acid that serves as a crucial inhibitory neurotransmitter in the brain, regulating various physiological functions. As a result of its potential benefits, GABA has gained substantial interest in the functional food and pharmaceutical industries. The enzyme responsible for GABA production is glutamic acid decarboxylase (GAD), which catalyzes the irreversible decarboxylation of glutamate. Understanding the crystal structure and catalytic mechanism of GAD is pivotal in advancing our knowledge of GABA production. This article provides an overview of GAD’s sources, structure, and catalytic mechanism, and explores strategies for enhancing GABA production through fermentation optimization, metabolic engineering, and genetic engineering. Furthermore, the effects of GABA on the physiological functions of animal organisms are also discussed. To meet the increasing demand for GABA, various strategies have been investigated to enhance its production, including optimizing fermentation conditions to facilitate GAD activity. Additionally, metabolic engineering techniques have been employed to increase the availability of glutamate as a precursor for GABA biosynthesis. By fine-tuning fermentation conditions and utilizing metabolic and genetic engineering techniques, it is possible to achieve higher yields of GABA, thus opening up new avenues for its application in functional foods and pharmaceuticals. Continuous research in this field holds immense promise for harnessing the potential of GABA in addressing various health-related challenges.

Nicotinamide mononucleotide (NMN), the direct precursor of nicotinamide adenine dinucleotide (NAD⁺), is involved in the regulation of many physiological and metabolic reactions in the body. NMN can indirectly affect cellular metabolic pathways, DNA repair, and senescence, while also being essential for maintaining tissues and dynamic metabolic equilibria, promoting healthy aging. Therefore, NMN has found many applications in the food, pharmaceutical, and cosmetics industries. At present, NMN synthesis strategies mainly include chemical synthesis and biosynthesis. Despite its potential benefits, the commercial production of NMN by organic chemistry approaches faces environmental and safety problems. With the rapid development of synthetic biology, it has become possible to construct microbial cell factories to produce NMN in a cost-effective way. In this review, we summarize the chemical and biosynthetic strategies of NMN, offering an overview of the recent research progress on host selection, chassis cell optimization, mining of key enzymes, metabolic engineering, and adaptive fermentation strategies. In addition, we also review the advances in the role of NMN in aging, metabolic diseases, and neural function. This review provides comprehensive technical guidance for the efficient biosynthesis of NMN as well as a theoretical basis for its application in the fields of food, medicine, and cosmetics.

Although Pichia pastoris was successfully used for heterologous gene expression for more than twenty years, many factors influencing protein expression remain unclear. Here, we optimized the expression of a thermophilic endoglucanase from Thermothielavioides terrestris (TtCel45A) for cost-effective production in Pichia pastoris. To achieve this, we established a multifactorial regulation strategy that involved selecting a genome-editing system, utilizing neutral loci, incorporating multiple copies of the heterologous expression cassette, and optimizing high-density fermentation for the co-production of single-cell protein (SCP). Notably, even though all neutral sites were used, there was still a slight difference in the enzymatic activity of heterologously expressed TtCel45A. Interestingly, the optimal gene copy number for the chromosomal expression of TtCel45A was found to be three, indicating limitations in translational capacity, post-translational processing, and secretion, ultimately impacting protein yields in P. pastoris. We suggest that multiple parameters might influence a kinetic competition between protein elongation and mRNA degradation. During high-density fermentation, the highest protein concentration and endoglucanase activity of TtCel45A with three copies reached 15.8 g/L and 9640 IU/mL, respectively. At the same time, the remaining SCP of P. pastoris exhibited a crude protein and amino acid content of up to 59.32% and 46.98%, respectively. These findings suggested that SCP from P. pastoris holds great promise as a sustainable and cost-effective alternative for meeting the global protein demand, while also enabling the production of thermophilic TtCel45A in a single industrial process.

Protein glycosylation is the most complex posttranslational modification process. Most cellulases from filamentous fungi contain N-glycosylation and O-glycosylation. Here, we discuss the potential roles of glycosylation on the characteristics and function of cellulases. The use of certain cultivation, inducer, and alteration of engineering glycosylation pathway can enable the rational control of cellulase glycosylation. Glycosylation does not occur arbitrarily and may tend to modify the 3D structure of cellulases by using specially distributed glycans. Therefore, glycoengineering should be considered comprehensively along with the spatial structure of cellulases. Cellulase glycosylation may be an evolution phenomenon, which has been considered as an economical way for providing different functions from identical proteins. In addition to gene and transcription regulations, glycosylation may be another regulation on the protein expression level. Enhanced understanding of the potential regulatory role of cellulase glycosylation will enable synthetic biology approaches for the development of commercial cellulase.