Biosynthesis of a Functional Fragment of Human Collagen II in Pichia pastoris.

Collagen II (COL2) is the major component of cartilage tissue and is widely applied in pharmaceuticals, food, and cosmetics. In this study, COL fragments were extracted from human COL2 for secretory expression in Pichia pastoris. Three variants were successfully secreted by shake flask cultivation with a yield of 73.3-100.7 mg/L. The three COL2 variants were shown to self-assemble into triple-helix at 4 °C and capable of forming higher order assembly of nanofiber and hydrogel. The bioactivities of the COL2 variants were validated, showing that sample 205 exhibited the best performance for inducing fibroblast differentiation and cell migration. Meanwhile, sample 205 and 209 exhibited higher capacity for inducing in vitro blood clotting than commercial mouse COL1. To overexpress sample 205, the expression cassettes were constructed with different promoters and signal peptides, and the fermentation condition was optimized, obtaining a yield of 172 mg/L for sample 205. Fed-batch fermentation was carried out using a 5 L bioreactor, and the secretory protease Pep4 was knocked out to avoid sample degradation, finally obtaining a yield of 3.04 g/L. Here, a bioactive COL2 fragment was successfully identified and can be overexpressed in P. pastoris; the variant may become a potential biomaterial for skin care.

Highly Efficient Biosynthesis of Rebaudioside M8 through Structure-Guided Engineering of Glycosyltransferase UGT94E13.

Given the low-calorie, high-sweetness characteristics of steviol glycosides (SGs), developing SGs with improved taste profiles is a key focus. Rebaudioside M8 (Reb M8), a novel non-natural SG derivative obtained through glycosylation at the C-13 position of rebaudioside D (Reb D) using glycosyltransferase UGT94E13, holds promise for further development due to its enhanced sweetness. However, the low catalytic activity of UGT94E13 hampers further research and commercialization. This study aimed to improve the enzymatic activity of UGT94E13 through semirational design, and a variant UGT94E13-F169G/I185G was obtained with the catalytic activity improved by 13.90 times. A cascade reaction involving UGT94E13-F169G/I185G and sucrose synthase AtSuSy was established to recycle uridine diphosphate glucose, resulting in an efficient preparation of Reb M8 with a yield of 98%. Moreover, according to the analysis of the distances between the substrate Reb D and enzymes as well as between Reb D and the glucose donor through molecular dynamics simulations, it is found that the positive effect of shortening the distance on glycosylation reaction activity accounts for the improved catalytic activity of UGT94E13-F169G/I185G. Therefore, this study addresses the bottleneck in the efficient production of Reb M8 and provides a foundation for its widespread application in the food industry.

Structural Insights into the Catalytic Cycle of Inulin Fructotransferase: From Substrate Anchoring to Product Releasing.

Carbohydrate degradation is crucial for living organisms due to their essential functions in providing energy and composing various metabolic pathways. Nevertheless, in the catalytic cycle of polysaccharide degradation, the details of how the substrates bind and how the products release need more case studies. Here, we choose an inulin fructotransferase (SpIFTase) as a model system, which can degrade inulin into functionally difructose anhydride I. At first, the crystal structures of SpIFTase in the absence of carbohydrates and complex with fructosyl-nystose (GF4), difructose anhydride I, and fructose are obtained, giving the substrate trajectory and product path of SpIFTase, which are further supported by steered molecular dynamics simulations (MDSs) along with mutagenesis. Furthermore, structural topology variations at the active centers of inulin fructotransferases are suggested as the structural base for product release, subsequently proven by substitution mutagenesis and MDSs. Therefore, this study provides a case in point for a deep understanding of the catalytic cycle with substrate trajectory and product path.

Biosynthesis of Natural and Unnatural Perylenequinones for Drug Development.

A wide range of perylenequinones (PQs) with diverse structures and versatile bioactivities have long been isolated, positioning them as highly promising agents for photodynamic therapy (PDT). However, the lack of an efficient and cost-effective method to obtain these compounds and to introduce structural diversity and complexity currently hinders their further research and application. In this concept, we present a comprehensive overview of the advancements in the biosynthetic pathways of natural PQs based on their structural classification, and also summarize recent progress in the biosynthesis of natural PQs and derivatives. These pioneering efforts may pave the way for structure modification and large-scale bioproduction of natural and unnatural PQs through synthetic biology strategies to promote their drug development.

Discovery of a Fungal P450 with an Unusual Two-Step Mechanism for Constructing a Bicyclo[3.2.2]nonane Skeleton.

The successful biomimetic or chemoenzymatic synthesis of target natural products (NPs) and their derivatives relies on enzyme discovery. Herein, we discover a fungal P450 BTG5 that can catalyze the formation of a bicyclo[3.2.2]nonane structure through an unusual two-step mechanism of dimerization and cyclization in the biosynthesis of beticolin 1, whose bicyclo[3.2.2]nonane skeleton connects an anthraquinone moiety and a xanthone moiety. Further investigation reveals that BTG5-T318 not only determines the substrate selectivity but also alters the catalytic reactions, which allows the separation of the reaction to two individual steps, thereby understanding its catalytic mechanism. It reveals that the first heterodimerization undergoes the common oxidation process for P450s, while the second uncommon formal redox-neutral cyclization step is proved as a redox-mediated reaction, which has never been reported. Therefore, this work advances our understanding of P450-catalyzed reactions and paves the way for expansion of the diversity of this class of NPs through synthetic biology.

Engineered CBEs based on Macaca fascicularis A3A with improved properties for precise genome editing.

Cytidine deaminase defines the properties of cytosine base editors (CBEs) for C-to-T conversion. Replacing the cytidine deaminase rat APOBEC1 (rA1) in CBEs with a human APOBEC3A (hA3A) improves CBE properties. However, the potential CBE application of macaque A3A orthologs remains undetermined. Our current study develops and evaluates engineered CBEs based on Macaca fascicularis A3A (mA3A). Here, we demonstrate that BE4-mA3A and its RNA-editing-derived variants exhibit improved CBE properties, except for DNA off-target activity, compared to BE3-rA1 and BE4-rA1. Unexpectedly, deleting Ser-Val-Arg (SVR) in BE4-mA3A dramatically reduces DNA and RNA off-target activities and improves editing accuracy, with on-target efficiency unaffected. In contrast, a chimeric BE4-hA3A-SVR+ shows editing efficiency increased by about 50%, with other properties unaffected. Our findings demonstrate that mA3A-based CBEs could provide prototype options with advantages over rA1- and hA3A-based CBEs for further optimization, highlighting the importance of the SVR motif in defining CBE intrinsic properties.

Selective synthesis of rebaudioside M2 through structure-guided engineering of glycosyltransferase UGT94D1.

Rebaudioside M2 (Reb M2), a novel steviol glycoside derivative, has limited industrial applications due to its low synthetic yield and selectivity. Herein, we identify UGT94D1 as a selective glycosyltransferase for rebaudioside D (Reb D), leading to the production of a mono ß-1,6-glycosylated derivative, Reb M2. A variant UGT94D1-F119I/D188P was developed through protein engineering. This mutant exhibited a 6.33-fold improvement in catalytic efficiency, and produced Reb M2 with 92% yield. Moreover, molecular dynamics simulations demonstrated that UGT94D1-F119I/D188P exhibited a shorter distance between the nucleophilic oxygen (OH6) of the substrate Reb D and uridine diphosphate glucose, along with an increased Ophosphate-C1-Oacceptor angle, thus improving the catalytic activity of the enzyme. Therefore, this study provides an efficient method for the selective synthesis of Reb M2 and paves the way for its applications in various fields.

High-Level De Novo Production of (2S)-Eriodictyol in Yarrowia Lipolytica by Metabolic Pathway and NADPH Regeneration Engineering.

(2S)-Eriodictyol, a polyphenolic flavonoid, has found widespread applications in health supplements and food additives. However, the limited availability of plant-derived (2S)-eriodictyol cannot meet the market demand. Microbial production of (2S)-eriodictyol faces challenges, including the low catalytic efficiency of flavone 3'-hydroxylase/cytochrome P450 reductase (F3'H/CPR), insufficient precursor supplementation, and inadequate NADPH regeneration. This study systematically engineered Yarrowia lipolytica for high-level (2S)-eriodictyol production. In doing this, the expression of F3'H/CPR was balanced, and the supply of precursors was enhanced by relieving feedback inhibition of the shikimate pathway, promoting fatty acid ß-oxidation, and increasing the copy number of synthetic pathway genes. These strategies, combined with NADPH regeneration, achieved an (2S)-eriodictyol titer of 423.6 mg/L. Finally, in fed-batch fermentation, a remarkable 6.8 g/L (2S)-eriodictyol was obtained, representing the highest de novo microbial titer reported to date and paving the way for industrial production.

Modular assembly of an artificially concise biocatalytic cascade for the manufacture of phenethylisoquinoline alkaloids.

Plant-derived alkaloids are an important class of pharmaceuticals. However, they still rely on phytoextraction to meet their diverse market demands. Since multistep biocatalytic cascades have begun to revolutionize the manufacture of natural or unnatural products, to address the synthetic challenges of alkaloids, herein we establish an artificially concise four-enzyme biocatalytic cascade with avoiding plant-derived P450 modification for synthesizing phenethylisoquinoline alkaloids (PEIAs) after enzyme discovery and enzyme engineering. Efficient biosynthesis of diverse natural and unnatural PEIAs is realized from readily available substrates. Most importantly, the scale-up preparation of the colchicine precursor (S)-autumnaline with a high titer is achieved after replacing the rate-limiting O-methylation by the plug-and-play strategy. This study not only streamlines future engineering endeavors for colchicine biosynthesis, but also provides a paradigm for constructing more artificial biocatalytic cascades for the manufacture of diverse alkaloids through synthetic biology.

Synthetic Biology-based Construction of Unnatural Perylenequinones with Improved Photodynamic Anticancer Activities.

The construction of structural complexity and diversity of natural products is crucial for drug discovery and development. To overcome high dark toxicity and poor photostability of natural photosensitizer perylenequinones (PQs) for photodynamic therapy, herein, we aim to introduce the structural complexity and diversity to biosynthesize the desired unnatural PQs in fungus Cercospora through synthetic biology-based strategy. Thus, we first elucidate the intricate biosynthetic pathways of class B PQs and reveal how the branching enzymes create their structural complexity and diversity from a common ancestor. This enables the rational reprogramming of cercosporin biosynthetic pathway in Cercospora to generate diverse unnatural PQs without chemical modification. Among them, unnatural cercosporin A displays remarkably low dark toxicity and high photostability with retention of great photodynamic anticancer and antimicrobial activities. Moreover, it is found that, unlike cercosporin, unnatural cercosporin A could be selectively accumulated in cancer cells, providing potential targets for drug development. Therefore, this work provides a comprehensive foundation for preparing unnatural products with customized functions through synthetic biology-based strategies, thus facilitating drug discovery pipelines from nature.

Photoenzymatic Enantioselective Synthesis of Oxygen-Containing Benzo-Fused Heterocycles.

New-to-nature biocatalysis in organic synthesis has recently emerged as a green and powerful strategy for the preparation of valuable chiral products, among which chiral oxygen-containing benzo-fused heterocycles are important structural motifs in pharmaceutical industry. However, the asymmetric synthesis of these compounds through radical-mediated methods is challenging. Herein, a novel asymmetric radical-mediated photoenzymatic synthesis strategy is developed to realize the efficient enantioselective synthesis of oxygen-containing benzo-fused heterocycles through structure-guided engineering of a flavin-dependent 'ene'-reductase GluER. It shows that variant GluER-W100H could efficiently produce various benzoxepinones, chromanone and indanone with different benzo-fused rings in high yields with great stereoselectivities under visible light. Moreover, these results are well supported by mechanistic experiments, revealing that this photoenzymatic process involves electron donor-acceptor complex formation, single electron transfer and hydrogen atom transfer. Therefore, we provide an alternative green approach for efficient chemoenzymatic synthesis of important chiral skeletons of bioactive pharmaceuticals.

Structural Insight into the Catalytic Mechanisms of an L-Sorbosone Dehydrogenase.

L-Sorbosone dehydrogenase (SNDH) is a key enzyme involved in the biosynthesis of 2-keto-L-gulonic acid , which is a direct precursor for the industrial scale production of vitamin C. Elucidating the structure and the catalytic mechanism is essential for improving SNDH performance. By solving the crystal structures of SNDH from Gluconobacter oxydans WSH-004, a reversible disulfide bond between Cys295 and the catalytic Cys296 residues is discovered. It allowed SNDH to switch between oxidation and reduction states, resulting in opening or closing the substrate pocket. Moreover, the Cys296 is found to affect the NADP+ binding pose with SNDH. Combining the in vitro biochemical and site-directed mutagenesis studies, the redox-based dynamic regulation and the catalytic mechanisms of SNDH are proposed. Moreover, the mutants with enhanced activity are obtained by extending substrate channels. This study not only elucidates the physiological control mechanism of the dehydrogenase, but also provides a theoretical basis for engineering similar enzymes.

Designing a cercosporin-bioinspired bifunctional algicide with flocculation and photocatalysis for efficiently controlling harmful cyanobacterial blooms.

Harmful cyanobacterial blooms (HCBs) are spreading in freshwater ecosystems worldwide, adversely affecting drinking water supplies, aquatic production, recreational and tourism activities. Therefore, the efficient and environmentally friendly method is still of interest to be developed to effectively control HCBs. Inspired by the excellent algicidal activity of cercosporin (CP), a novel metal-free algaecide SiO2@EDU@CP (EDU, N-ethyl-N'-(3-dimethylaminopropyl)urea) with flocculation and photoremoval functions, was successfully designed and prepared in one-step to simultaneously introduce CP and EDU on SiO2 nanoparticles. It could rapidly form algae flocs in 20 min with 97.1% flocculation rate, and remove Microcystis aeruginosa within 12 h with 91.0% algicidal rate under 23 W compact fluorescent light irradiation without any leaked CP detected. Additionally, odorant ß-cyclocitral and toxin microcystin-LR were both photodegraded after treatment of SiO2@EDU@CP. Further mechanistic studies showed that the introduction of EDU significantly reversed the zeta potential of SiO2-COOH to achieve the flocculation through neutral charge, and the photophysical characterization of SiO2@EDU@CP revealed the improved charge separation ability to generate reactive oxygen species. More importantly, the utility of SiO2@EDU@CP was well demonstrated by its effectiveness for algae from Taihu Lake under natural sunlight and inability to regrow after treatment. This study not only establishes a bifunctional algicide SiO2@EDU@CP to efficiently control HCBs, but also provides design possibilities to develop more novel and efficient algicides for the better control of practical HCBs.

Enhanced production of aspochalasin D through genetic engineering of Aspergillus flavipes.

Aspochalasin D (AD) belongs to the polyketide-amino acid hybrid natural products with anti-cancer, anti-bacterial, and anti-fouling bioactivities. However, the low production limits its further application. In this study, AD was separated and identified from Aspergillus flavipes 3.17641. Next, besides the optimization of culture conditions using a single-factor experiment and response surface methodology, metabolic engineering was employed to increase the AD production. It shows that the deletion of the shunt gene aspoA and overexpression of the pathway-specific regulator aspoG significantly improve the AD production. Its production reached to 812.1 mg/L under the optimized conditions, with 18.5-fold increase. Therefore, this study not only provides a general method for improving the production of similar natural products in other fungi, but also enables the further biological function development of AD in agriculture and pharmaceutical. KEY POINTS: • The Aspochalasin D (AD) production was improved by optimizing culture conditions. • The deletion of the shunt gene aspoA increased the AD production. • Overexpression of the pathway regulator aspoG further improved the AD production.

Highly efficient synthesis of mono-ß-1,6-Glucosylated Rebaudioside A derivative catalyzed by glycosyltransferase YjiC.

Steviol glycosides have attracted great interest because of their high levels of sweetness and safety, and absence of calories. Improvement of their sensory qualities via glycosylation modification by glycosyltransferase is a research hotspot. In this study, YjiC, a uridine diphosphate-dependent glycosyltransferase from Bacillus subtilis 168, was found with the ability to glycosylate rebaudioside A (Reb A) to produce a novel mono ß-1, 6-glycosylated Reb A derivative rebaudioside L2 (Reb L2). It has an improved sweetness compared with Reb A. Next, a cascade reaction was established by combining YjiC with sucrose synthase AtSuSy from Arabidopsis thaliana for scale-up preparation of Reb L2. It shows that Reb L2 (30.94 mg/mL) could be efficiently synthesized with an excellent yield of 91.34% within 12 h. Therefore, this study provides a potential approach for the production and application of new steviol glycoside Reb L2, expanding the scope of steviol glycosides.

Structural analysis of an anthrol reductase inspires enantioselective synthesis of enantiopure hydroxycycloketones and ß-halohydrins.

Asymmetric reduction of prochiral ketones, particularly, reductive desymmetrization of 2,2-disubstituted prochiral 1,3-cyclodiketones to produce enantiopure chiral alcohols is challenging. Herein, an anthrol reductase CbAR with the ability to accommodate diverse bulky substrates, like emodin, for asymmetric reduction is identified. We firstly solve crystal structures of CbAR and CbAR-Emodin complex. It reveals that Tyr210 is critical for emodin recognition and binding, as it forms a hydrogen-bond interaction with His162 and π-π stacking interactions with emodin. This ensures the correct orientation for the stereoselectivity. Then, through structure-guided engineering, variant CbAR-H162F can convert various 2,2-disubstituted 1,3-cyclodiketones and α-haloacetophenones to optically pure (2S, 3S)-ketols and (R)-ß-halohydrins, respectively. More importantly, their stereoselectivity mechanisms are also well explained by the respective crystal structures of CbAR-H162F-substrate complex. Therefore, this study demonstrates that an in-depth understanding of catalytic mechanism is valuable for exploiting the promiscuity of anthrol reductases to prepare diverse enantiopure chiral alcohols.

Biosynthetic Pathways of Dimeric Natural Products Containing Bisanthraquinone and Related Xanthones.

Many dimeric natural products containing bisanthraquinone and related xanthones with diverse structures and versatile bioactivities have been isolated over the years. However, the complicated biosynthetic pathways of such natural products, which have remained elusive until recently, negatively impact their mass bioproduction and biosynthetic structural modification for drug discovery. In this concept, we summarize the recent progress in gene cluster mining and biosynthetic pathway elucidation of natural products containing bisanthraquinone and related xanthones. These pioneering works may pave the way for further biosynthetic pathway elucidation and structure modification of dimeric natural products through gene and protein engineering.

Manipulation of fungal cell wall integrity to improve production of fungal natural products.

Fungi, as an important industrial microorganism, play an essential role in the production of natural products (NPs) due to their advantages of utilizing cheap raw materials as substrates and strong protein secretion ability. Although many metabolic engineering strategies have been adopted to enhance the biosynthetic pathway of NPs in fungi, the fungal cell wall as a natural barrier tissue is the final and key step that affects the efficiency of NPs synthesis. To date, many important progresses have been achieved in improving the synthesis of NPs by regulating the cell wall structure of fungi. In this review, we systematically summarize and discuss various strategies for modifying the cell wall structure of fungi to improve the synthesis of NPs. At first, the cell wall structure of different types of fungi is systematically described. Then, strategies to disrupt cell wall integrity (CWI) by regulating the synthesis of cell wall polysaccharides and binding proteins are summarized, which have been applied to improve the synthesis of NPs. In addition, we also summarize the studies on the regulation of CWI-related signaling pathway and the addition of exogenous components for regulating CWI to improve the synthesis of NPs. Finally, we propose the current challenges and essential strategies to usher in an era of more extensive manipulation of fungal CWI to improve the production of fungal NPs.

Characterization of highly gelatinous patatin storage protein from Pichia pastoris.

Patatin is a useful plant protein with excellent gelation properties that could be used as a gelling agent in the food industry. However, the commercial production of patatin is limited because the traditional extraction methods are inefficient and time consuming. Production of patatin with gelation properties by microorganisms is a promising alternative route. In this study, 1424.5 mg/L patatin storage protein with great gelation properties could be obtained in a 5-L bioreactor after optimization of the signal peptide, the promoter, and the fed-batch process when a Pichia pastoris GS115, but not Escherichia coli, expression system was used. Compared with commercial potato-extracted patatins, P. pastoris-derived patatins showed better gelation properties, such as a lower gel-forming concentration and gelation temperature. In addition, the gel strength of P. pastoris-derived patatins was comparable with that of potato-extracted patatins. These results suggested that P. pastoris-derived patatins have the potential to replace current potato-derived ones, which are now widely used in plant-based meat products.

Stimulating fungal cell wall integrity by exogenous ß-glucanase to improve the production of fungal natural products.

The low production of natural products (NPs) is still the critical restrictive factor in exploiting their potential large-scale applications and a barrier to isolating and identifying other meaningful products. Given that the stimulation of cell wall integrity (CWI) has become a novel strategy to modulate the production of microbial natural products, herein, exogenous ß-glucanase treatment was developed as an external cell wall ß-glucan stress to stimulate the fungal CWI, and then to improve the production of fungal NPs. It was found that the production of fungal NPs cercosporin and sophorolipids, biosynthesized by Cercospora sp. and Starmerella bombicola, respectively, was significantly improved by the treatment of ß-glucanase under a controllable dose. Moreover, it demonstrated that ß-glucanase had an ability to stimulate fungal CWI through slight fungal superficial damage, thus facilitating the secretion of NPs. We expected that this easy-operating method to stimulate fungal CWI could be feasible to improve more fungal NPs production. KEY POINTS: • Exogenous ß-glucanase stimulated the fungal cell wall integrity • Changing fungal cell walls modulated natural product production • ß-glucanase with potential universal effects on more fungal natural products.