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[学术文献 ] Identification of Subfamily Specific Residues within Highly Active and Promiscuous Alcohol Dehydrogenases 进入全文
ACS Catalysis
Enzyme selection is an essential process in the biobased production of chemicals. It is essential to develop a method to extract yet unknown useful enzymes from protein databases. Enzymes that exhibit substrate promiscuity and high activity hold the potential to access unknown reactions and mediate known reactions with a higher performance. Herein, we propose and validate a principal component analysis (PCA)-based classification method, termed MUSASHI (MUltiple-Sequence Alignment-based protein Selection via clustering using HIgh-dimensional analysis), to identify subfamily-specific residues that are highly conserved among promiscuous alcohol dehydrogenase (ADH). Specifically, zinc-dependent ADH homologues retrieved from the protein database were classified into 9 groups, and according to PCA-based clustering, the activities of 18 ADHs, with representative enzymes from each group, were characterized. As a result, we identified two promiscuous ADH groups: Group 1 ADH, efficient with short-chain and aromatic aldehydes, and Group 3 ADH, efficient with aliphatic and aromatic ketones. Sequence feature analysis then revealed subfamily-specific residues, which are highly conserved only in promiscuous ADH Groups 1 and 3, with the potential to biosynthesize a wide spectrum of target compounds. Tatumella ptyseos ADH, identified from Group 1 of this study, showed higher isobutanol and 2-phenylethanol bioconversions than that of a conventional ADH (Ahr). These results indicate that the MUSASHI method for subfamily-specific residue identification can enable optimal enzyme selection from protein databases.
[学术文献 ] Enhancing the thermal stability and activity of the engineered self-sufficient P450SPα-SOX by switching the domains linker 进入全文
International Journal of Biological Macromolecules
This work reports on the engineering of the linker between P450 SPα (CYP152B1) and sarcosine oxidase (SOX), with the aim of enhancing the structural rigidity of the fusion protein (SPα-SOX) and study the effect on its stability and catalytic performance. Differential scanning calorimetry shows that the construct bearing the rigid linker (SPα-rigidSOX) results in a higher energy barrier to unfolding (765 kcal/mol) compared to the previous fusion system (SPα-flexible-SOX) (561 kcal/mol), as well as a Tonset above 50 °C. Furthermore, residual CO-binding after heat treatment was investigated for both the fusion systems, and a 5.7 °C increase of the T50 of SPα-rigid-SOX is shown. Interestingly, a stabilized P420 semifolded state of the SPα is also observed after SPα-rigid-SOX incubation at high temperature (40°). The two fusion systems were studied at high temperature for the turnover of lauric acid: SPα-rigid-SOX shows a 98 % conversion yield using 5 mM substrate compared to the 24 % conversion of SPαflexibleSOX when the catalysis is carried out at 40 °C. Finally, the activity of the two constructs was tested using styrene as a substrate, and three products of catalysis were observed: styrene oxide (85 %), phenylacetaldehyde (0–3 %) and 2-phenylpropenal (12–15 %). Interestingly, 2-phenylpropenal is observed for the first time and only for the fusion enzymes. Also in this case, SPα-rigid-SOX outperformed SPα-flexible-SOX with a 3-fold higher conversion yield. Overall, we demonstrate that the rigid linker improves the fusion enzyme thermal stability and catalytic performance, both at high temperature and in mild conditions, resulting also in the production of new molecules of biotechnological interest.
[学术文献 ] Discovery to Engineering of Mycotoxin Deoxynivalenol Degrading Enzymes Based on the Specialized Glyoxalase I 进入全文
Advanced Science
Deoxynivalenol (DON) is a mycotoxin that is omnipresent in food and feed. Therefore, this study has focused on discovery, molecular characterization, and engineering of DON degrading enzymes, based on a DON isomerizing enzyme (e.g., the specialized glyoxalase I from Gossypium raimondii (Gr-SPG)). A molecular phylogeny-based sequence and structure analysis elucidated the evolutionary trajectory of the DON degrading enzymes. Ancestral sequence reconstruction led to the generation of thermostable evolutionary intermediates of SPG (e.g., Anc216). Molecular modeling and consensus protein design allowed to understand the structure and function relationships and also identify the key conserved mutations that influence catalytic activity and thermostability. Ultimately, a highly active and thermostable SPG (e.g., a quintuple mutant of Anc216 (Anc216_M5)) was constructed from a newly discovered extant SPG enzyme (OR9). The Anc216_M5 exhibited a T5010 of 68 °C, which is 16.3 °C higher than that of the wild-type enzyme. Furthermore, the engineered enzyme showed 40% greater DON degrading activity than OR9, which is significantly higher than that of Gr-SPG. Therefore, it is assumed that Anc216_M5 is promising as a DON-detoxifying biocatalyst.
[学术文献 ] Chemoenzymatic platform with coordinated cofactor self-circulation for lignin valorization 进入全文
Nature Synthesis
Lignin valorization is vital for achieving economically viable and sustainable lignocellulosic biorefineries. However, the value-added utilization of lignin constituents is hampered by the requirement of expensive cofactors and low conversions. Here, by integrating coenzyme regeneration and cell-free expression, we report an in vitro multienzyme-coordinated expression with cofactor self-circulation (iMECS) strategy to achieve efficient lignin-to-molecule conversion. The iMECS system established a catalytic bridge with high atom economy for converting lignin waste into valuable aromatic compounds. Curcumin, vanillin and raspberry ketone were efficiently synthesized in a coenzyme-free manner, with a conversion of over 90%. We also demonstrated the flexibility of the iMECS platform, through which a wide array of phenylpropanoids could be easily obtained by enzyme swapping and pathway extension. By integrating chemical depolymerization with the iMECS system, lignin-rich agricultural waste was directly converted into valuable chemicals, and the overall catalytic efficiency was enhanced by up to 48-fold compared with the reported titres. This efficient, general platform can facilitate the utilization of lignocellulosic biomass, thereby promoting zero-waste biorefineries for a more sustainable future.
[学术文献 ] An mRNA-display derived cyclic peptide scaffold reveals the substrate binding interactions of an N-terminal cysteine oxidase 进入全文
Nature Communications
N-terminal cysteine oxidases (NCOs) act as enzymatic oxygen (O2) sensors, coordinating cellular changes to hypoxia in animals and plants. They regulate the O2-dependent stability of proteins bearing an N-terminal cysteine residue through the N-degron pathway. Despite their important role in hypoxic adaptation, which renders them potential therapeutic and agrichemical targets, structural information on NCO substrate binding remains elusive. To overcome this challenge, we employed a unique strategy by which a cyclic peptide inhibitor of the mammalian NCO, 2-aminoethanethiol dioxygenase (ADO), was identified by mRNA display and used as a scaffold to graft substrate moieties. This allowed the determination of two substrate analogue-bound crystal structures of ADO. Key binding interactions were revealed, including bidentate coordination of the N-terminal residue at the metal cofactor. Subsequent structure guided mutagenesis identified aspartate-206 as an essential catalytic residue, playing a role in reactive oxygen intermediate orientation or stabilisation. These findings provide fundamental information on ADO substrate interactions, which can elucidate enzyme mechanism and act as a platform for chemical discovery.
[前沿资讯 ] 天津工业生物技术研究所通过电击诱变和代谢工程改造创制高产虾青素的裂殖壶菌细胞工厂 进入全文
中国科学院天津工业生物技术研究所
虾青素（astaxanthin）是一种强效抗氧化剂，在医药、保健品、化妆品等领域具有广泛的应用。在虾青素单酯的各种分子构型中，DHA与虾青素形成的单酯具有最高的生物利用度和稳定性。裂殖壶菌（Schizochytrium sp）因其高油脂含量及产量，并含有EPA、DHA等多不饱和脂肪酸，以及少量角鲨烯、虾青素等类胡萝卜素而受到广泛关注。然而天然的裂殖壶菌虾青素产量极低，无法满足工业化需求。 近日，中国科学院天津工业生物技术研究所李德茂研究员带领的工业生物系统工程研究团队成功构建出一株高产虾青素的裂殖壶菌细胞工厂。研究团队通过电穿孔诱变技术强化MVA途径前体供应，成功构建Schizochytrium sp. AST32底盘菌株，使虾青素含量从7.56μg/g显著提升至55.17μg/g。基于该突变株，通过理性设计策略进一步优化虾青素生物合成途径，在Schizochytrium sp. AST32中同时表达五个关键基因（idi、crtE、crtIBY、crtZ、crtW），使虾青素含量跃升至374.85μg/g。进一步，应用PLIN融合蛋白技术将虾青素合成模块定向锚定于细胞脂滴，使虾青素产量提升至400.38μg/g。经发酵工艺优化后，最终获得虾青素产量峰值29.53mg/L（1913.07μg/g，较初始突变株提升169.72倍），同时维持DHA产量3.83g/L（占总脂质含量的45%）。该研究为推进裂殖壶菌虾青素生物合成的商业化应用奠定了关键技术基础。
