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Background: Genome-scale metabolic models (GEMs) serve as effective tools for understanding cellular phenotypes and predicting engineering targets in the development of industrial strain. Enzyme-constrained genome-scale metabolic models (ecGEMs) have emerged as a valuable advancement, providing more accurate predictions and unveiling new engineering targets compared to models lacking enzyme constraints. In 2022, a stoichiometric GEM, iDL1450, was reconstructed for the industrially significant fungus Myceliophthora thermophila. To enhance the GEM's performance, an ecGEM was developed for M. thermophila in this study. Results: Initially, the model iDL1450 underwent refinement and updates, resulting in a new version named iYW1475. These updates included adjustments to biomass components, correction of gene-protein-reaction (GPR) rules, and a consensus on metabolites. Subsequently, the first ecGEM for M. thermophila was constructed using machine learning-based kcat data predicted by TurNuP within the ECMpy framework. During the construction, three versions of ecGEMs were developed based on three distinct kcat collection methods, namely AutoPACMEN, DLKcat and TurNuP. After comparison, the ecGEM constructed using TurNuP-predicted kcat values performed better in several aspects and was selected as the definitive version of ecGEM for M. thermophila (ecMTM). Comparing ecMTM to iYW1475, the solution space was reduced and the growth simulation results more closely resembled realistic cellular phenotypes. Metabolic adjustment simulated by ecMTM revealed a trade-off between biomass yield and enzyme usage efficiency at varying glucose uptake rates. Notably, hierarchical utilization of five carbon sources derived from plant biomass hydrolysis was accurately captured and explained by ecMTM. Furthermore, based on enzyme cost considerations, ecMTM successfully predicted reported targets for metabolic engineering modification and introduced some new potential targets for chemicals produced in M. thermophila. Conclusions: In this study, the incorporation of enzyme constraint to iYW1475 not only improved prediction accuracy but also broadened the model's applicability. This research demonstrates the effectiveness of integrating of machine learning-based kcat data in the construction of ecGEMs especially in situations where there is limited measured enzyme kinetic parameters for a specific organism. [ABSTRACT FROM AUTHOR]

The α-1 antitrypsin Z-mutant protein (ATZ) is the primary cause of α-1 antitrypsin deficiency (AATD). Studying the ubiquitination modification and degradation of ATZ protein is importance for developing treatments for AATD. STUB1 is an important E3 ubiquitin ligase that regulates ubiquitination modification of various proteins. However, whether STUB1 in involved in the ubiquitination modification of ATZ has not been fully elucidated. In this study, the ATZ and STUB1 coding genes were first cloned into the pET28a plasmid, constructing 2 protein expression plasmids. The recombinant plasmids were then transferred into the Escherichia coli for expression. With the optimization of induction temperature and IPTG dosage, the recombinant proteins were successfully expressed. The target proteins were then efficiently purified from cell lysates using metal-chelating affinity chromatography, and the accuracy of the amino acid sequence was verified through protein mass spectrometry analysis. Using the purified ATZ and STUB1, we established an in vitro ubiquitination reaction system. Experimental results showed that, in the presence of ATP, E1 ubiquitin-activating enzyme, and E2 ubiquitin-conjugating enzyme, STUB1 catalyzed the ubiquitination modification of ATZ. This study provides a method for obtaining the ATZ protein in vitro , elucidates the mechanism of STUB1 mediating ATZ ubiquitination, thereby advancing our understanding of the intracellular degradation mechanism of the α-1 antitrypsin Z-mutant.

Plant biomass represents a vast resource of carbon. In China, it is estimated that 1 billion tons of biomass is available each year. The conversion of these biomass resources into bioethanol or other bio-based chemicals, if fully commercialized, may reduce at least 200 million tons of crude oil import. Therefore, bioethanol and bulk chemicals are the core components of the biomanufacturing using plant biomass as carbon sources. Since the foundation of Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences (TIB, CAS), we have proposed a strategy of "two replacements and one upgrade". Utilizing renewable carbon resources instead of non-renewable petrochemical resources to produce bulk chemicals is included in our strategy. It is a long-term effort for TIB to develop plant biomass biomanufacturing to produce renewable chemicals. Continuous and systematic research was carried out in these two fields, and significant progress has been made in the past 10 years since the foundation of TIB. Here we review the progress of TIB in this field, mainly focusing on fungal system, including the mechanism of cellulose degradation by filamentous fungi and the strategy of consolidated bioprocessing of biomass. Based on this, malic acid, fuel ethanol and other bulk chemicals were produced through one-step conversion of biomass. Besides, the commercial processes for production of bulk chemicals such as succinic and lactic acid from renewable carbon resources, which were developed by TIB, were also be discussed. These examples clearly demonstrated that bulk chemicals can be obtained from biomass instead of from petroleum. Research on plant biomass biotransformation and renewable chemicals production in TIB has provided an alternative route for the development of low-carbon bioeconomy in China, and will contribute to the goal of carbon neutralization of China.

Utilization of carbon dioxide (CO 2 ) is a huge challenge for global sustainable development. Biological carbon fixation occurs in nature, but the low energy efficiency and slow speed hamper its commercialization. Physical-chemical carbon fixation is efficient, but relies on high energy consumption and often generates unwanted by-products. Combining the advantages of biological, physical and chemical technologies for efficient utilization of CO 2 remains to be an urgent scientific and technological challenge to be addressed. Here, based on the development of Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences in the past decade, we summarize the important progress in the design and construction of functional parts, pathways and systems for artificial bioconversion of carbon dioxide, including the breakthrough on the artificial synthesis of starch from CO 2 . Moreover, we prospect how to further develop the technologies for artificial bioconversion of carbon dioxide. These progress and perspectives provide new insight for achieving the goal of "carbon peaking and carbon neutrality".