Rational construction of genome-minimized Streptomyces host for the expression of secondary metabolite gene clusters.

Streptomyces offer a wealth of naturally occurring compounds with diverse structures, many of which possess significant pharmaceutical values. However, new product exploration and increased yield of specific compounds in Streptomyces have been technically challenging due to their slow growth rate, complex culture conditions and intricate genetic backgrounds. In this study, we screened dozens of Streptomyces strains inhabiting in a plant rhizosphere for fast-growing candidates, and further employed CRISPR/Cas-based engineering techniques for stepwise refinement of a particular strain, Streptomyces sp. A-14 that harbors a 7.47 Mb genome. After strategic removal of nonessential genomic regions and most gene clusters, we reduced its genome size to 6.13 Mb, while preserving its growth rate to the greatest extent. We further demonstrated that cleaner metabolic background of this engineered strain was well suited for the expression and characterization of heterologous gene clusters, including the biosynthetic pathways of actinorhodin and polycyclic tetramate macrolactams. Moreover, this streamlined genome is anticipated to facilitate directing the metabolic flux towards the production of desired compounds and increasing their yields.

Hybrid Cas12a Variants with Relaxed PAM Requirements Expand Genome Editing Compatibility.

Cas12a is a widely used programmable nuclease for genome editing across a variety of organisms, but its application is limited by its PAM recognition restriction. To alleviate these PAM constraints, protein engineering efforts have been applied to expand the PAM recognition range. In this study, we designed and constructed 990 synthetic hybrid Cas12a chimeras through domain shuffling and screened an efficient hybrid Cas12a (ehCas12a) that could recognize a broad range PAM of 5'-TYYN-3' (Y is T or C and N is A, T, C, or G). Furthermore, we constructed an ehCas12a variant, ehCas12a RRVR (T167R/N572R/K578V/N582R), with expanded PAM preference to 5'-TNYN, TWRV-3' (W is A or T, R is A or G, and V is A, C, or G), which can efficiently recognize -2* A/G PAMs that are barely recognized by Cas12a-type proteins and their mutants. Finally, we demonstrated that the DNase-inactivated ehCas12a RRVR base editor (dehCas12a RRVR-BE) was capable of targeting noncanonical PAMs in vivo and disease-related loci for potential therapeutic applications. Overall, our findings highlight the modular design and reconfiguration of Cas proteins for enhanced functionality.

Synthesis of Trifluoromethylated Monoterpenes by an Engineered Cytochrome P450.

Protein engineering of cytochrome P450s has enabled these biocatalysts to promote a variety of abiotic reactions beyond nature's repertoire. Integrating such non-natural transformations with microbial biosynthetic pathways could allow sustainable enzymatic production of modified natural product derivatives. In particular, trifluoromethylation is a highly desirable modification in pharmaceutical research due to the positive effects of the trifluoromethyl group on drug potency, bioavailability, and metabolic stability. This study demonstrates the biosynthesis of non-natural trifluoromethyl-substituted cyclopropane derivatives of natural monoterpene scaffolds using an engineered cytochrome P450 variant, P411-PFA. P411-PFA successfully catalyzed the transfer of a trifluoromethyl carbene from 2-diazo-1,1,1-trifluoroethane to the terminal alkenes of several monoterpenes, including L-carveol, carvone, perilla alcohol, and perillartine, to generate the corresponding trifluoromethylated cyclopropane products. Furthermore, integration of this abiotic cyclopropanation reaction with a reconstructed metabolic pathway for L-carveol production in Escherichia coli enabled one-step biosynthesis of a trifluoromethylated L-carveol derivative from limonene precursor. Overall, amalgamating synthetic enzymatic chemistry with established metabolic pathways represents a promising approach to sustainably produce bioactive natural product analogs.

Recent Advances in the Biosynthesis of Natural Sugar Substitutes in Yeast.

Natural sugar substitutes are safe, stable, and nearly calorie-free. Thus, they are gradually replacing the traditional high-calorie and artificial sweeteners in the food industry. Currently, the majority of natural sugar substitutes are extracted from plants, which often requires high levels of energy and causes environmental pollution. Recently, biosynthesis via engineered microbial cell factories has emerged as a green alternative for producing natural sugar substitutes. In this review, recent advances in the biosynthesis of natural sugar substitutes in yeasts are summarized. The metabolic engineering approaches reported for the biosynthesis of oligosaccharides, sugar alcohols, glycosides, and rare monosaccharides in various yeast strains are described. Meanwhile, some unresolved challenges in the bioproduction of natural sugar substitutes in yeast are discussed to offer guidance for future engineering.

Mechanistic investigation of a D to N mutation in DAHP synthase that dictates carbon flux into the shikimate pathway in yeast.

3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS) is a key enzyme in the shikimate pathway for the biosynthesis of aromatic compounds. L-Phe and L-Tyr bind to the two main DAHPS isoforms and inhibit their enzyme activities, respectively. Synthetic biologists aim to relieve such inhibitions in order to improve the productivity of aromatic compounds. In this work, we reported a point mutant of yeast DHAPS, Aro3D154N, which retains the wild type enzyme activity but converts it highly inert to the inhibition by L-Phe. The Aro3 crystal structure along with the molecular dynamics simulations analysis suggests that the D154N mutation distant from the inhibitor binding cavity may reduce the binding affinity of L-Phe. Growth assays demonstrated that substitution of the conserved D154 with asparagine suffices to relieve the inhibition of L-Phe on Aro3, L-Tyr on Aro4, and the inhibitions on their corresponding homologues from diverse yeasts. The importance of our discovery is highlighted by the observation of 29.1% and 43.6% increase of yield for the production of tyrosol and salidroside respectively upon substituting ARO3 with ARO3D154N. We anticipate that this allele would be used broadly to increase the yield of various aromatic products in metabolically diverse microorganisms.

Anaerobic purinolytic enzymes enable dietary purine clearance by engineered gut bacteria.

Uric acid, the end product of purine degradation, causes hyperuricemia and gout, afflicting hundreds of millions of people. The debilitating effects of gout are exacerbated by dietary purine intake, and thus a potential therapeutic strategy is to enhance purine degradation in the gut microbiome. Aerobic purine degradation involves oxidative dearomatization of uric acid catalyzed by the O2-dependent uricase. The enzymes involved in purine degradation in strictly anaerobic bacteria remain unknown. Here we report the identification and characterization of these enzymes, which include four hydrolases belonging to different enzyme families, and a prenyl-flavin mononucleotide-dependent decarboxylase. Introduction of the first two hydrolases to Escherichia coli Nissle 1917 enabled its anaerobic growth on xanthine as the sole nitrogen source. Oral supplementation of these engineered probiotics ameliorated hyperuricemia in a Drosophila melanogaster model, including the formation of renal uric acid stones and a shortened lifespan, providing a route toward the development of purinolytic probiotics.

Yeast: A platform for the production of L -tyrosine derivatives.

L -Tyrosine derivatives are widely applied in the pharmaceutical, food, and chemical industries. Their production is mainly confined to chemical synthesis and plant extract. Microorganisms, as cell factories, exhibit promising advantages for valuable chemical production to fulfill the increase in the demand of global markets. Yeast has been used to produce natural products owing to its robustness and genetic maneuverability. Focusing on the progress of yeast cell factories for the production of L -tyrosine derivatives, we summarized the emerging metabolic engineering approaches in building L -tyrosoine-overproducing yeast and constructing cell factories of three typical chemicals and their derivatives: tyrosol, p-coumaric acid, and L -DOPA. Finally, the challenges and opportunities of L -tyrosine derivatives production in yeast cell factories were also discussed.

Structural basis of the substrate recognition and inhibition mechanism of Plasmodium falciparum nucleoside transporter PfENT1.

By lacking de novo purine biosynthesis enzymes, Plasmodium falciparum requires purine nucleoside uptake from host cells. The indispensable nucleoside transporter ENT1 of P. falciparum facilitates nucleoside uptake in the asexual blood stage. Specific inhibitors of PfENT1 prevent the proliferation of P. falciparum at submicromolar concentrations. However, the substrate recognition and inhibitory mechanism of PfENT1 are still elusive. Here, we report cryo-EM structures of PfENT1 in apo, inosine-bound, and inhibitor-bound states. Together with in vitro binding and uptake assays, we identify that inosine is the primary substrate of PfENT1 and that the inosine-binding site is located in the central cavity of PfENT1. The endofacial inhibitor GSK4 occupies the orthosteric site of PfENT1 and explores the allosteric site to block the conformational change of PfENT1. Furthermore, we propose a general "rocker switch" alternating access cycle for ENT transporters. Understanding the substrate recognition and inhibitory mechanisms of PfENT1 will greatly facilitate future efforts in the rational design of antimalarial drugs.

High-Level Production of Hydroxytyrosol in Engineered Saccharomyces cerevisiae.

Hydroxytyrosol (HT) is a valuable aromatic compound with numerous applications. Herein, we enabled the efficient and scalable de novo HT production in engineered Saccharomyces cerevisiae (S. cerevisiae) from glucose. Starting from a tyrosol-overproducing strain, six HpaB/HpaC combinations were investigated, and the best catalytic performance was acquired with HpaB from Pseudomonas aeruginosa (PaHpaB) and HpaC from Escherichia coli (EcHpaC), resulting in 425.7 mg/L HT in shake flasks. Next, weakening the tryptophan biosynthetic pathway through downregulating the expression of TRP2 (encoding anthranilate synthase) further improved the HT titer by 27.2% compared to the base strain. Moreover, the cytosolic NADH supply was improved through introducing the feedback-resistant mutant of the TyrA (the NAD+-dependent chorismate mutase/prephenate dehydrogenase, TyrA*) from E. coli, which further increased the HT titer by 36.9% compared to the base strain. The best performing strain was obtained by optimizing the biosynthesis of HT in S. cerevisiae through a screening for an effective HpaB/HpaC combination, biosynthetic flux rewiring, and cofactor engineering, which enabled the titer of HT reaching 1120.0 mg/L in the shake flask. Finally, the engineered strain produced 6.97 g/L of HT by fed-batch fermentation, which represents the highest titer for de novo HT biosynthesis in microorganisms reported to date.

Metabolite-based biosensors for natural product discovery and overproduction.

Natural products derived from bacterial secondary metabolites contribute greatly to the pharmaceutical industry. Heterologous expression of natural product biosynthetic pathways can remarkably improve the yield of target products and lead to the discovery of numerous derivatives. Therefore, high-throughput analytical methods are urgently needed for the detection of natural products. Biosensors allow fast, real-time detection and efficient screening. With the growth of in-depth knowledge of biosensors, biosensors with high efficiency and specificity are exploited for broader applications. Here, we summarized how biosensors targeting different metabolites were constructed and optimized and the applications of metabolite-based biosensors in heterologous bacterial hosts. Finally, we prospected the future development of biosensors, including combinations with other advanced technologies, to solve the challenges hampering wider applications.

Research progress of pathway and genome evolution in microbes.

Microbes can produce valuable natural products widely applied in medicine, food and other important fields. Nevertheless, it is usually challenging to achieve ideal industrial yields due to low production rate and poor toxicity tolerance. Evolution is a constant mutation and adaptation process used to improve strain performance. Generally speaking, the synthesis of natural products in microbes is often intricate, involving multiple enzymes or multiple pathways. Individual evolution of a certain enzyme often fails to achieve the desired results, and may lead to new rate-limiting nodes that affect the growth of microbes. Therefore, it is inevitable to evolve the biosynthetic pathways or the whole genome. Here, we reviewed the pathway-level evolution including multi-enzyme evolution, regulatory elements engineering, and computer-aided engineering, as well as the genome-level evolution based on several tools, such as genome shuffling and CRISPR/Cas systems. Finally, we also discussed the major challenges faced by in vivo evolution strategies and proposed some potential solutions.

[Recent advances in CRISPR-related transposable elements].

A new wave of research has been inspired by the CRISPR-Cas system with respect to their application in genome editing. The CRISPR-Cas system can not only be applied in gene knockout and insertion, but also be used in base editing, transcriptional regulation and recombination of gene clusters. However, the low efficiency of homology-directed repair (HDR) limits its application. Unlike the CRISPR-Cas system, mobile genetic elements (MGE) can insert DNA fragments into cell chromosomes without the aid of HDR. Recently, it is reported that CRISPR-related transposable elements can guide targeted DNA insertion. Their transposition mechanisms and reprogramming abilities have brought novel opportunities to the development of this field. This review summarized the research progress and application development of natural CRISPR-related transposable elements in recent years, as well as the applications of fused dCas9-transposase. It proposed the application prospects and potential challenges of CRISPR-related transposable elements in the future, which provided a reference for the development direction of gene editing tools.

Harnessing the Endogenous 2µ Plasmid of Saccharomyces cerevisiae for Pathway Construction.

pRS episomal plasmids are widely used in Saccharomyces cerevisiae, owing to their easy genetic manipulations and high plasmid copy numbers (PCNs). Nevertheless, their broader application is hampered by the instability of the pRS plasmids. In this study, we designed an episomal plasmid based on the endogenous 2µ plasmid with both improved stability and increased PCN, naming it p2µM, a 2µ-modified plasmid. In the p2µM plasmid, an insertion site between the REP1 promoter and RAF1 promoter was identified, where the replication (ori) of Escherichia coli and a selection marker gene of S. cerevisiae were inserted. As a proof of concept, the tyrosol biosynthetic pathway was constructed in the p2µM plasmid and in a pRS plasmid (pRS423). As a result, the p2µM plasmid presented lower plasmid loss rate than that of pRS423. Furthermore, higher tyrosol titers were achieved in S. cerevisiae harboring p2µM plasmid carrying the tyrosol pathway-related genes. Our study provided an improved genetic manipulation tool in S. cerevisiae for metabolic engineering applications, which may be widely applied for valuable product biosynthesis in yeast.

Endogenous 2µ Plasmid Editing for Pathway Engineering in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, conventional 2µ-plasmid based plasmid (pC2µ, such as pRS425) have been widely adopted in pathway engineering for multi-copy overexpression of key genes. However, the loss of partition and copy number control elements of yeast endogenous 2µ plasmid (pE2µ) brings the issues concerning plasmid stability and copy number of pC2µ, especially in long-term fermentation. In this study, we developed a method based on CRISPR/Cas9 to edit pE2µ and built the pE2µ multi-copy system by insertion of the target DNA element and elimination of the original pE2µ plasmid. The resulting plasmid pE2µRAF1 and pE2µREP2 demonstrated higher copy number and slower loss rate than a pC2µ control plasmid pRS425RK, when carrying the same target gene. Then, moving the essential gene TPI1 (encoding triose phosphate isomerase) from chromosome to pE2µRAF1 could increase the plasmid viability to nearly 100% and further increase the plasmid copy number by 73.95%. The expression using pE2µ multi-copy system demonstrated much smaller cell-to-cell variation comparing with pC2µ multi-copy system. With auxotrophic complementation of TPI1, the resulting plasmid pE2µRT could undergo cultivation of 90 generations under non-selective conditions without loss. Applying pE2µ multi-copy system for dihydroartemisinic acid (DHAA) biosynthesis, the production of DHAA was increased to 620.9 mg/L at shake-flask level in non-selective rich medium. This titer was 4.73-fold of the strain constructed based on pC2µ due to the more stable pE2µ plasmid system and with higher plasmid copy number. This study provides an improved expression system in yeast, and set a promising platform to construct biosynthesis pathway for valuable products.

Recent Advances in Silent Gene Cluster Activation in Streptomyces.

Natural products (NPs) are critical sources of drug molecules for decades. About two-thirds of natural antibiotics are produced by Streptomyces. Streptomyces have a large number of secondary metabolite biosynthetic gene clusters (SM-BGCs) that may encode NPs. However, most of these BGCs are silent under standard laboratory conditions. Hence, activation of these silent BGCs is essential to current natural products discovery research. In this review, we described the commonly used strategies for silent BGC activation in Streptomyces from two aspects. One focused on the strategies applied in heterologous host, including methods to clone and reconstruct BGCs along with advances in chassis engineering; the other focused on methods applied in native host which includes engineering of promoters, regulatory factors, and ribosomes. With the metabolic network being elucidated more comprehensively and methods optimized more high-thoroughly, the discovery of NPs will be greatly accelerated.

IsPETase Is a Novel Biocatalyst for Poly(ethylene terephthalate) (PET) Hydrolysis.

Poly(ethylene terephthalate) (PET) is one of the most widely used synthetic polyesters, but also a major cause of plastic pollution. Because the chemical degradation of PET would be uneconomical and rather burdensome, considerable efforts have been devoted to exploring enzymatic processes for the disposal of PET waste. Many PET-hydrolyzing enzymes have been reported in recent decades, some of which demonstrate excellent potential for industrial applications. This review sets out to summarize the state of investigation into IsPETase, a cutinase-like enzyme from Ideonella sakaiensis possessing ability to degrade crystalline PET, and to gain further insight into the structure-function relationship of IsPETase. Benefiting from the continuing identification of novel cutinase-like proteins and growing availability of the engineered IsPETase, we may anticipate future developments in this type of enzyme would generate suitable biocatalyst for industrial use.

Label-free proteomic dissection on dptP-deletion mutant uncovers dptP involvement in strain growth and daptomycin tolerance of Streptomyces roseosporus.

Daptomycin (DAP) is a novel microbial lipopeptide antibiotic synthesized by the DAP biosynthetic gene cluster dpt of Streptomyces roseosporus (S. roseosporus). DptP gene locates upstream of dpt and confers DAP resistance to Streptomyces ambofaciens (S. ambofaciens). So far, the biological functions of dptP gene for S. roseosporus growth are still completely uncovered. We performed label-free quantification proteomic dissections with loss- and gain-of-function experiments to decipher dptP-involved functions. Deletion of dptP gene activated energy metabolism and metabolism of secondary metabolites pathways and enhanced the transcription levels and protein abundance of key members of the dpt cluster. Whereas dptP deletion inhibited transport/signal transduction and drug resistance pathways and protein abundance of cell division-relative proteins, subsequently decreased mycelia cell growth rate. S. roseosporus strain with dptP deletion was more sensitive to DAP treatment compared to the wild type. In contrast, overexpression of dptP gene decreased transcription levels of DAP biosynthetic genes and enhanced growth rate of Streptomcyes strain upon elevated culture temperature and DAP supplementation. Taken together, dptP gene contributes to Streptomcyes primary growth under elevated temperature and DAP treatment, whereas it plays negative roles on metabolism of secondary metabolites and transcription of DAP biosynthetic genes.

Multi-modular engineering of Saccharomyces cerevisiae for high-titre production of tyrosol and salidroside.

Tyrosol and its glycosylated product salidroside are important ingredients in pharmaceuticals, nutraceuticals and cosmetics. Despite the ability of Saccharomyces cerevisiae to naturally synthesize tyrosol, high yield from de novo synthesis remains a challenge. Here, we used metabolic engineering strategies to construct S. cerevisiae strains for high-level production of tyrosol and salidroside from glucose. First, tyrosol production was unlocked from feedback inhibition. Then, transketolase and ribose-5-phosphate ketol-isomerase were overexpressed to balance the supply of precursors. Next, chorismate synthase and chorismate mutase were overexpressed to maximize the aromatic amino acid flux towards tyrosol synthesis. Finally, the competing pathway was knocked out to further direct the carbon flux into tyrosol synthesis. Through a combination of these interventions, tyrosol titres reached 702.30 ± 0.41 mg l-1 in shake flasks, which were approximately 26-fold greater than that of the WT strain. RrU8GT33 from Rhodiola rosea was also applied to cells and maximized salidroside production from tyrosol in S. cerevisiae. Salidroside titres of 1575.45 ± 19.35 mg l-1 were accomplished in shake flasks. Furthermore, titres of 9.90 ± 0.06 g l-1 of tyrosol and 26.55 ± 0.43 g l-1 of salidroside were achieved in 5 l bioreactors, both are the highest titres reported to date. The synergistic engineering strategies presented in this study could be further applied to increase the production of high value-added aromatic compounds derived from the aromatic amino acid biosynthesis pathway in S. cerevisiae.

Structural and biochemical studies of the glycosyltransferase Bs-YjiC from Bacillus subtilis.

Glycosylation possess prominent biological and pharmacological significance in natural product and drug candidate synthesis. The glycosyltransferase YjiC, discovered from Bacillus subtilis (Bs-YjiC), shows potential applications in drug development due to its wide substrate spectrums. In order to elucidate its catalytic mechanism, we solved the crystal structure of Bs-YjiC, demonstrating that Bs-YjiC adopts a typical GT-B fold consisting of a flexible N-domain and a relatively rigid C-domain. Structural analysis coupled with site-directed mutagenesis studies revealed that site Ser277 was critical for Nucleoside Diphosphate (NDP) recognition, while Glu317, Gln318, Ser128 and Ser129 were crucial for glycosyl moiety recognition. Our results illustrate the structural basis for acceptor promiscuity in Bs-YjiC and provide a starting point for further protein engineering of Bs-YjiC in industrial and pharmaceutical applications.

Synthesis and evaluation of new compounds bearing 3-(4-aminopiperidin-1-yl)methyl magnolol scaffold as anticancer agents for the treatment of non-small cell lung cancer via targeting autophagy.

Magnolol and honokiol are the two major active ingredients with similar structure and anticancer activity from traditional Chinese medicine Magnolia officinalis, and honokiol is now in a phase I clinical trial (CTR20170822) for advanced non-small cell lung cancer (NSCLC). In search of potent lead compounds with better activity, our previous study has demonstrated that magnolol derivative C2, 3-(4-aminopiperidin-1-yl)methyl magnolol, has better activity than honokiol. Here, based on the core of 3-(4-aminopiperidin-1-yl)methyl magnolol, we synthesized fifty-one magnolol derivatives. Among them, compound 30 exhibited the most potent antiproliferative activities on H460, HCC827, H1975 cell lines with the IC50 values of 0.63-0.93 µM, which were approximately 10- and 100-fold more potent than those of C2 and magnolol, respectively. Besides, oral administration of 30 and C2 on an H460 xenograft model also demonstrated that 30 has better activity than C2. Mechanism study revealed that 30 induced G0/G1 phase cell cycle arrest, apoptosis and autophagy in cancer cells. Moreover, blocking autophagy by the autophagic inhibitor enhanced the anticancer activity of 30in vitro and in vivo, suggesting autophagy played a cytoprotective role on 30-induced cancer cell death. Taken together, our study implied that compound 30 combined with autophagic inhibitor could be another choice for NSCLC treatment in further investigation.