Chemical manufacturing, which relies on fossil as feedstocks, often encounters challenges such as poor economic performance, non-renewable resources depletion and environmental pollution. In contrast, biomanufacturing represents a novel approach to green chemistry by utilizing enzymes or cellular entities as the core tool for chemical production. Microbial cell factories (MCFs), powered by the advances of synthetic biology, has emerged as an environmentally friendly precept for biomanufacturing fuels, materials and chemicals (Jiang et al., 2020; Marsafari et al., 2020; Montano Lopez et al., 2021; Yang et al., 2022). In recent years, microorganisms have been engineered to produce chemicals from renewable feedstocks, such as Clostridium species for the production of butanol (Lutke-Eversloh and Bahl, 2011), Escherichia coli for the production of PHAs (Zhuang et al., 2014), Saccharomyces cerevisiae for the production of opioid pain-relieving drugs thebaine (Galanie et al., 2015). However, many highly valuable chemicals cannot be efficiently produced through MCFs in industrial scales, because of the inability of MCFs to meeting stringent titer, rate, and yield (TRY) requirements.

Many metabolic engineering strategies including promoter engineering (Yamaguchi et al., 2013), transcription factor engineering (Courchesne et al., 2009), cofactor engineering (Chen et al., 2014b), compartmentalization engineering (Lee et al., 2012), and modular pathway engineering (Biggs et al., 2014), have been used to improve the TRY of MCFs. Recently, the nonconventional yeast Ogataea polymorpha was metabolically rewired by optimizing the mevalonate pathway, enhancing the supply of NADPH and acetyl-coA, and downregulating the competition pathways, and thus produced 509 mg/L and 4.7 g/L of β-elemene under batch fermentation and feed-batch fermentation, respectively (Ye et al., 2023). However, most metabolic enzymes exhibit low catalytic activity, poor stability, and unexpected substrate/product inhibition under industrial conditions (Foo et al., 2012; Leonard et al., 2010), resulting in the decreased TRY of MCFs. For example, in the assembled L-homophenylalanine pathway, TipheDH was one of rate-limiting enzymes due to low catalytic activity. To deal with this issue, the specific activity of TipheDH was engineered by combining semi-saturated mutation with high-throughput screening, and thus its specific activity was improved by 82%, leading to L-homophenylalanine production up to 100.9 g/L. Productivity in MCFs depends on metabolic protein status, which is largely determined by the rational control of target proteins to enable biological systems to have the desired phenotype. Therefore, protein status engineering, which consists of protein engineering, protein modification and protein assembly, plays an important role in improving the efficiency of MCFs.

Protein status engineering is primarily underpinned by the strategic amalgamation of genetic and protein engineering techniques. This fusion serves as a transformative approach, endowing proteins with specific attributes and practical utilities by tailoring their structure, function, and expression. Based on this, protein status engineering has been used to regulate target proteins to enable biological system with desired phenotypes which is especially important for enhancing cellular synthetic capacity. Recently, many studies focus on elucidating the metabolic efficiency at protein status engineering, including redesigning glycolyl-CoA carboxylase (GCC) active-site for improving catalytic efficiency to match the tartronyl-CoA (TaCo) pathway (Scheffen et al., 2021), engineering quorum sensing protein degradation circuit for controlling Erg9 degradation to produce α-farnesene (Yang et al., 2021), using metabolic enzymes to form multienzyme compartmentalization by liquid-liquid phase separation (LLPS) to enhance terpene biosynthesis (Guo et al., 2022). In summary, the field of protein status engineering encompasses precise control over protein structure and function, intricate cellular processes, and molecular interactions. Thus, it is necessary for the development of novel tools and methods for customized design and fine-tuning of proteins.

In this review, we focus on status of protein to further increase the production capacity of MCFs, which can be integrated with synthetic biological strategies and exploitation of microbial physiological mechanisms (Fig. 1). Engineering protein status mainly focuses on the protein engineering for boosting catalytic capacity, protein modification for regulating microbial metabolic capacity, and protein assembly for enhancing microbial synthetic capacity. Protein status engineering can optimize the allocation of metabolic resources in MCFs. Finally, we discuss the future challenges and prospects in the engineering of protein status to improve the production performance of MCFs.