CsCCD2 Access Tunnel Design for a Broader Substrate Profile in Crocetin Production.

Crocetin, a high-value apocarotenoid in saffron, is widely applied to the fields of food and medicine. However, the existing method of obtaining crocetin through large-scale cultivation is far from meeting the market demand. Microbial synthesis of crocetin is a potential alternative to traditional resources, and it is found that carotenoid cleavage dioxygenase (CCD) is the critical enzyme to synthesize crocetin. So, in this study, we used "hybrid-tunnel" engineering to obtain variants of Crocus sativus-derived CsCCD2, essential for zeaxanthin conversion into crocetin, with a broader substrate specificity and higher catalytic efficiency. Variants including S323A, with a lower charge bias and a larger tunnel size than the wild-type, showed a 5-fold higher crocetin titer in yeast-based fermentations. S323A could also convert the ß-carotene substrate to crocetin dialdehyde and exhibited a 12.83-fold greater catalytic efficiency (kcat/Km) toward zeaxanthin than the wild-type in vitro. This strategy enabled the production of 107 mg/L crocetin in 5 L fed-batch fermentation, higher than that previously reported. Our findings demonstrate that engineering access tunnels to expand the substrate profile by in silico protein design represents a viable strategy to refine the catalytic properties of enzymes across a range of applications.

Compartmentalized Reconstitution of Post-squalene Pathway for 7-Dehydrocholesterol Overproduction in Saccharomyces cerevisiae.

7-Dehydrocholesterol (7-DHC) is the direct precursor to manufacture vitamin D3. Our previous study has achieved 7-DHC synthesis in Saccharomyces cerevisiae based on the endogenous post-squalene pathway. However, the distribution of post-squalene enzymes between the endoplasmic reticulum (ER) and lipid bodies (LD) might raise difficulties for ERG proteins to catalyze and deliver sterol intermediates, resulting in unbalanced metabolic flow and low product yield. Herein, we intended to rearrange the subcellular location of post-squalene enzymes to alleviate metabolic bottleneck and boost 7-DHC production. After identifying the location of DHCR24 (C-24 reductase, the only heterologous protein for 7-DHC biosynthesis) on ER, all the ER-located enzymes were grouped into four modules: ERG1/11/24, ERG25/26/27, ERG2/3, and DHCR24. These modules attempted to be overexpressed either on ER or on LDs. As a result, expression of LD-targeted DHCR24 and ER-located ERG1/11/24 could promote the conversion efficiency among the sterol intermediates to 7-DHC, while locating module ERG2/3 into LDs improved the whole metabolic flux of the post-squalene pathway. Coexpressing LD-targeted ERG2/3 and DHCR24 (generating strain SyBE_Sc01250035) improved 7-DHC production from 187.7 to 308.2 mg/L at shake-flask level. Further expressing ER-targeted module ERG1/11/24 in strain SyBE_Sc01250035 dramatically reduced squalene accumulation from 620.2 mg/L to the lowest level (by 93.8%) as well as improved 7-DHC production to the highest level (to 342.2 mg/L). Then targeting module ERG25/26/27 to LDs further increased 7-DHC titer to 360.6 mg/L, which is the highest shake-flask level production for 7-DHC ever reported. Our study not only proposes and further proves the concept of pathway compartmentalized reconstitution to regulate metabolic flux but also provides a promising chassis to produce other steroidal compounds through the post-squalene pathway.

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.

NVD-BM-mediated genetic biosensor triggers accumulation of 7-dehydrocholesterol and inhibits melanoma via Akt1/NF-ĸB signaling.

Aberrant activation of the cholesterol biosynthesis supports tumor cell growth. In recent years, significant progress has been made by targeting rate-limiting enzymes in cholesterol biosynthesis pathways to prevent carcinogenesis. However, precise mechanisms behind cholesterol degradation in cancer cells have not been comprehensively investigated. Here, we report that codon optimization of the orthologous cholesterol 7-desaturase, NVD-BM from Bombyx mori, significantly slowed melanoma cell proliferation and migration, and inhibited cancer cell engraftment in nude mice, by converting cholesterol to toxic 7-dehydrocholesterol. Based on these observations, we established a synthetic genetic circuit to induce melanoma cell regression by sensing tumor specific signals in melanoma cells. The dual-input signals, RELA proto-oncogene (RELA) and signal transducer and activator of transcription 1 (STAT1), activated NVD-BM expression and repressed melanoma cell proliferation and migration. Mechanically, we observed that NVD-BM decreased Akt1-ser473 phosphorylation and inhibited cytoplasmic RELA translocation. Taken together, NVD-BM was identified as a tumor suppressor in malignant melanoma, and we established a dual-input biosensor to promote cancer cell regression, via Akt1/NF-κB signaling. Our results demonstrate the potential therapeutic effects of cholesterol 7-desaturase in melanoma metabolism, and provides insights for genetic circuits targeting 7-dehydrocholesterol accumulation in tumors.

Exploring Catalysis Specificity of Phytoene Dehydrogenase CrtI in Carotenoid Synthesis.

Carotenoids, a variety of natural products, have significant pharmaceutical and commercial potential. Phytoene dehydrogenase (CrtI) is the rate-limit enzyme for carotenoid synthesis, whose catalysis specificity results in various carotenoids. However, the structural characteristics of CrtI for controlling the catalysis specificity on dehydrogenation steps are still unclear, which limited the development of CrtI function. Here we confirmed two mutation sites H136 and H453 in the mutant library of CrtI from Blakeslea trispora, which markedly regulated catalytic specificity. Interestingly, the sequence alignment features at H136 and H453 were consistent with the phylogenetic analysis of CrtI families. Subsequently, the functions of saturated mutants at H136 and H453 were clustered by principal component analysis (PCA) and k-means. According to the clustering results, diversiform mutants with specific dehydrogenation function provided important application value for carotenoid product customization. Meanwhile, this study also enriched the theory of enzyme evolution and guided the functional development of enzymes.

Metabolic Engineering of Saccharomyces cerevisiae for Enhanced Dihydroartemisinic Acid Production.

Direct bioproduction of DHAA (dihydroartemisinic acid) rather than AA (artemisinic acid), as suggested by previous work would decrease the cost of semi-biosynthesis artemisinin by eliminating the step of initial hydrogenation of AA. The major challenge in microbial production of DHAA is how to efficiently manipulate consecutive key enzymes ADH1 (artemisinic alcohol dehydrogenase), DBR2 [artemisinic aldehyde Δ11(13) reductase] and ALDH1 (aldehyde dehydrogenase) to redirect metabolic flux and elevate the ratio of DHAA to AA (artemisinic acid). Herein, DHAA biosynthesis was achieved in Saccharomyces cerevisiae by introducing a series of heterologous enzymes: ADS (amorpha-4,11-diene synthase), CYP71AV1 (amorphadiene oxidase), ADH1, DBR2 and ALDH1, obtaining initial DHAA/AA ratio at 2.53. The flux toward DHAA was enhanced by pairing fusion proteins DBR2-ADH1 and DBR2-ALDH1, leading to 1.75-fold increase in DHAA/AA ratio (to 6.97). Moreover, to promote the substrate preference of ALDH1 to dihydroartemisinic aldehyde (the intermediate for DHAA synthesis) over artemisinic aldehyde (the intermediate for AA synthesis), two rational engineering strategies, including downsizing the active pocket and enhancing the stability of enzyme/cofactor complex, were proposed to engineer ALDH1. It was found that the mutant H194R, which showed better stability of the enzyme/NAD+ complex, obtained the highest DHAA to AA ratio at 3.73 among all the mutations. Then the mutant H194R was incorporated into above rebuilt fusion proteins, resulting in the highest ratio of DHAA to AA (10.05). Subsequently, the highest DHAA reported titer of 1.70 g/L (DHAA/AA ratio of 9.84) was achieved through 5 L bioreactor fermentation. The study highlights the synergy of metabolic engineering and protein engineering in metabolic flux redirection to get the most efficient product to the chemical process, and simplified downstream conversion process.

Metabolic engineering of Methylobacterium extorquens AM1 for the production of butadiene precursor.

BACKGROUND: Butadiene is a platform chemical used as an industrial feedstock for the manufacture of automobile tires, synthetic resins, latex and engineering plastics. Currently, butadiene is predominantly synthesized as a byproduct of ethylene production from non-renewable petroleum resources. Although the idea of biological synthesis of butadiene from sugars has been discussed in the literature, success for that goal has so far not been reported. As a model system for methanol assimilation, Methylobacterium extorquens AM1 can produce several unique metabolic intermediates for the production of value-added chemicals, including crotonyl-CoA as a potential precursor for butadiene synthesis. RESULTS: In this work, we focused on constructing a metabolic pathway to convert crotonyl-CoA into crotyl diphosphate, a direct precursor of butadiene. The engineered pathway consists of three identified enzymes, a hydroxyethylthiazole kinase (THK) from Escherichia coli, an isopentenyl phosphate kinase (IPK) from Methanothermobacter thermautotrophicus and an aldehyde/alcohol dehydrogenase (ADHE2) from Clostridium acetobutylicum. The Km and kcat of THK, IPK and ADHE2 were determined as 8.35 mM and 1.24 s-1, 1.28 mM and 153.14 s-1, and 2.34 mM and 1.15 s-1 towards crotonol, crotyl monophosphate and crotonyl-CoA, respectively. Then, the activity of one of rate-limiting enzymes, THK, was optimized by random mutagenesis coupled with a developed high-throughput screening colorimetric assay. The resulting variant (THKM82V) isolated from over 3000 colonies showed 8.6-fold higher activity than wild-type, which helped increase the titer of crotyl diphosphate to 0.76 mM, corresponding to a 7.6% conversion from crotonol in the one-pot in vitro reaction. Overexpression of native ADHE2, IPK with THKM82V under a strong promoter mxaF in M. extorquens AM1 did not produce crotyl diphosphate from crotonyl-CoA, but the engineered strain did generate 0.60 µg/mL of intracellular crotyl diphosphate from exogenously supplied crotonol at mid-exponential phase. CONCLUSIONS: These results represent the first step in producing a butadiene precursor in recombinant M. extorquens AM1. It not only demonstrates the feasibility of converting crotonol to key intermediates for butadiene biosynthesis, it also suggests future directions for improving catalytic efficiency of aldehyde/alcohol dehydrogenase to produce butadiene precursor from methanol.

Metabolic engineering of Saccharomyces cerevisiae for 7-dehydrocholesterol overproduction.

BACKGROUND: 7-Dehydrocholesterol (7-DHC) has attracted increasing attentions due to its great medical value and the enlarging market demand of its ultraviolet-catalyzed product vitamin D3. Microbial production of 7-DHC from simple carbon has been recognized as an attractive complement to the traditional sources. Even though our previous work realized 7-DHC biosynthesis in Saccharomyces cerevisiae, the current productivity of 7-DHC is still too low to satisfy the demand of following industrialization. As increasing the compatibility between heterologous pathway and host cell is crucial to realize microbial overproduction of natural products with complex structure and relative long pathway, in this study, combined efforts in tuning the heterologous Δ24-dehydrocholesterol reductase (DHCR24) and manipulating host cell were applied to promote 7-DHC accumulation. RESULTS: In order to decouple 7-DHC production with cell growth, inducible GAL promoters was employed to control 7-DHC synthesis. Meanwhile, the precursor pool was increased via overexpressing all the mevalonate (MVA) pathway genes (ERG10, ERG13, tHMG1, ERG12, ERG8, ERG19, IDI1, ERG20). Through screening DHCR24s from eleven tested sources, it was found that DHCR24 from Gallus gallus (Gg_DHCR24) achieved the highest 7-DHC production. Then 7-DHC accumulation was increased by 27.5% through stepwise fine-tuning the transcription level of Gg_DHCR24 in terms of altering its induction strategy, integration position, and the used promoter. By blocking the competitive path (ΔERG6) and supplementing another copy of Gg_DHCR24 in locus ERG6, 7-DHC accumulation was further enhanced by 1.07-fold. Afterward, 7-DHC production was improved by 48.3% (to 250.8 mg/L) by means of deleting NEM1 that was involved in lipids metabolism. Eventually, 7-DHC production reached to 1.07 g/L in 5-L bioreactor, which is the highest reported microbial titer as yet known. CONCLUSIONS: Combined engineering of the pathway and the host cell was adopted in this study to boost 7-DHC output in the yeast. 7-DHC titer was stepwise improved by 26.9-fold compared with the starting strain. This work not only opens large opportunities to realize downstream de novo synthesis of other steroids, but also highlights the importance of the combinatorial engineering of heterologous pathway and host to obtain microbial overproduction of many other natural products.

Identification and manipulation of a novel locus to improve cell tolerance to short-chain alcohols in Escherichia coli.

Escherichia coli KO11 is a popular ethanologenic strain, but is more sensitive to ethanol than other producers. Here, an ethanol-tolerant mutant EM was isolated from ultraviolet mutagenesis library of KO11. Comparative genomic analysis added by piecewise knockout strategy and complementation assay revealed EKO11_3023 (espA) within the 36.6-kb deletion from KO11 was the only locus responsible for ethanol sensitivity. Interestingly, when espA was deleted in strain W (the parent strain of KO11), ethanol tolerance was dramatically elevated to the level of espA-free hosts [e.g., MG1655 and BL21(DE3)]. And overexpression of espA in strains MG1655 and BL21(DE3) led to significantly enhanced ethanol sensitivity. In addition to ethanol, deletion of espA also improved cell tolerance to other short-chain (C2-C4) alcohols, including methanol, isopropanol, n-butanol, isobutanol and 2-butanol. Therefore, espA was responsible for short-chain alcohol sensitivity of W-strains compared to other cells, which provides a potential engineering target for alcohols production.

Chassis and key enzymes engineering for monoterpenes production.

Microbial production of monoterpenes is often limited by their cytotoxicity and in vivo conversion. Therefore, alleviating cytotoxicity and reducing conversion by chassis engineering are highly desirable. On the other hand, engineering key enzymes is also critical for improving monoterpenes production through facilitating the biosynthesis process. Here we critically review recent advances in cytotoxicity alleviation, reducing in vivo conversion, selecting geranyl diphosphate synthase and engineering monoterpene synthases. These achievements would lead to the development of superior chassis with improved tolerance to cytotoxicity and rationally tailored metabolites profiles to improve titer, yield and productivity for the production of monoterpenes by microbial cells.

Manipulation of GES and ERG20 for geraniol overproduction in Saccharomyces cerevisiae.

Manipulation of monoterpene synthases to maximize flux towards targeted products from GPP (geranyl diphosphate) is the main challenge for heterologous monoterpene overproduction, in addition to cell toxicity from compounds themselves. In our study, by manipulation of the key enzymes geraniol synthase (GES) and farnesyl diphosphate synthase (Erg20), geraniol (a valuable acyclic monoterpene alcohol) overproduction was achieved in Saccharomyces cerevisiae with truncated 3-hydroxy-3-methylglutaryl-coenzyme reductase (tHMGR) and isopentenyl diphosphate isomerase (IDI1) overexpressed. The expressions of all above engineered genes were under the control of Gal promoter for alleviating product toxicity. Geraniol production varied from trace amount to 43.19mg/L (CrGES, GES from Catharanthus roseus) by screening of nine GESs from diverse species. Further through protein structure analysis and site-directed mutation in CrGES, it was firstly demonstrated that among the high-conserved amino acid residues located in active pocket, Y436 and D501 with strong affinity to diphosphate function group, were critical for the dephosphorylation (the core step for geraniol formation). Moreover, the truncation position of the transit peptide from the N-terminus of CrGES was found to influence protein expression and activity significantly, obtaining a titer of 191.61mg/L geraniol in strain with CrGES truncated at S43 (t3CrGES). Furthermore, directed by surface electrostatics distribution of t3CrGES and Erg20WW (Erg20F96W-N127W), co-expression of the reverse fusion of Erg20ww/t3CrGES and another copy of Erg20WW promoted the geraniol titer to 523.96mg/L at shakes flask level, due to enhancing GPP accessibility led by protein interaction of t3CrGES-Erg20WW and the free Erg20WW. Eventually, a highest reported titer of 1.68g/L geraniol in eukaryote cells was achieved in 2.0L fed-batch fermentation under carbon restriction strategy. Our research opens large opportunities for other microbial production of monoterpenes. It also sets a good reference for desired compounds overproduction in microorganisms in terms of manipulation of key enzymes by protein engineering and metabolic engineering.

Complex structure of the DNA-binding domain of AdpA, the global transcription factor in Streptomyces griseus, and a target duplex DNA reveals the structural basis of its tolerant DNA sequence specificity.

AdpA serves as the global transcription factor in the A-factor regulatory cascade, controlling the secondary metabolism and morphological differentiation of the filamentous bacterium Streptomyces griseus. AdpA binds to over 500 operator regions with the consensus sequence 5'-TGGCSNGWWY-3' (where S is G or C, W is A or T, Y is T or C, and N is any nucleotide). However, it is still obscure how AdpA can control hundreds of genes. To elucidate the structural basis of this tolerant DNA recognition by AdpA, we focused on the interaction between the DNA-binding domain of AdpA (AdpA-DBD), which consists of two helix-turn-helix motifs, and a target duplex DNA containing the consensus sequence 5'-TGGCGGGTTC-3'. The crystal structure of the AdpA-DBD-DNA complex and the mutant analysis of AdpA-DBD revealed its unique manner of DNA recognition, whereby only two arginine residues directly recognize the consensus sequence, explaining the strict recognition of G and C at positions 2 and 4, respectively, and the tolerant recognition of other positions of the consensus sequence. AdpA-DBD confers tolerant DNA sequence specificity to AdpA, allowing it to control hundreds of genes as a global transcription factor.

Purification, crystallization and preliminary X-ray analysis of the DNA-binding domain of AdpA, the central transcription factor in the A-factor regulatory cascade in the filamentous bacterium Streptomyces griseus, in complex with a duplex DNA.

Streptomyces griseus AdpA is the central transcription factor in the A-factor regulatory cascade and activates a number of genes that are required for both secondary metabolism and morphological differentiation, leading to the onset of streptomycin biosynthesis as well as aerial mycelium formation and sporulation. The DNA-binding domain of AdpA consists of two helix-turn-helix DNA-binding motifs and shows low nucleotide-sequence specificity. To reveal the molecular basis of the low nucleotide-sequence specificity, an attempt was made to obtain cocrystals of the DNA-binding domain of AdpA and several kinds of duplex DNA. The best diffracting crystal was obtained using a 14-mer duplex DNA with two-nucleotide overhangs at the 5'-ends. The crystal diffracted X-rays to 2.8 Šresolution and belonged to space group C222(1), with unit-cell parameters a = 76.86, b = 100.96, c = 101.25 Å. The Matthews coefficient (V(M) = 3.71 Å(3) Da(-1)) indicated that the crystal was most likely to contain one DNA-binding domain of AdpA and one duplex DNA in the asymmetric unit, with a solvent content of 66.8%.