Utilizing a cell-free protein synthesis platform for the biosynthesis of a natural product, caffeine.

Natural products are a valuable source of pharmaceuticals, providing a majority of the small-molecule drugs in use today. However, their production through organic synthesis or in heterologous hosts can be difficult and time-consuming. Therefore, to allow for easier screening and production of natural products, we demonstrated the use of a cell-free protein synthesis system to partially assemble natural products in vitro using S-Adenosyl Methionine (SAM)-dependent methyltransferase enzyme reactions. The tea caffeine synthase, TCS1, was utilized to synthesize caffeine within a cell-free protein synthesis system. Cell-free systems also provide the benefit of allowing the use of substrates that would normally be toxic in a cellular environment to synthesize novel products. However, TCS1 is unable to utilize a compound like S-adenosyl ethionine as a cofactor to create ethylated caffeine analogs. The automation and reduced metabolic engineering requirements of cell-free protein synthesis systems, in combination with other synthesis methods, may enable the more efficient generation of new compounds. Graphical Abstract.

An NmrA-like enzyme-catalysed redox-mediated Diels-Alder cycloaddition with anti-selectivity.

The Diels-Alder cycloaddition is one of the most powerful approaches in organic synthesis and is often used in the synthesis of important pharmaceuticals. Yet, strictly controlling the stereoselectivity of the Diels-Alder reactions is challenging, and great efforts are needed to construct complex molecules with desired chirality via organocatalysis or transition-metal strategies. Nature has evolved different types of enzymes to exquisitely control cyclization stereochemistry; however, most of the reported Diels-Alderases have been shown to only facilitate the energetically favourable diastereoselective cycloadditions. Here we report the discovery and characterization of CtdP, a member of a new class of bifunctional oxidoreductase/Diels-Alderase, which was previously annotated as an NmrA-like transcriptional regulator. We demonstrate that CtdP catalyses the inherently disfavoured cycloaddition to form the bicyclo[2.2.2]diazaoctane scaffold with a strict α-anti-selectivity. Guided by computational studies, we reveal a NADP+/NADPH-dependent redox mechanism for the CtdP-catalysed inverse electron demand Diels-Alder cycloaddition, which serves as the first example of a bifunctional Diels-Alderase that utilizes this mechanism.

Multiplex Base-Editing Enables Combinatorial Epigenetic Regulation for Genome Mining of Fungal Natural Products.

Genome mining of cryptic natural products (NPs) remains challenging, especially in filamentous fungi, owing to their complex genetic regulation. Increasing evidence indicates that several epigenetic modifications often act cooperatively to control fungal gene transcription, yet the ability to predictably manipulate multiple genes simultaneously is still largely limited. Here, we developed a multiplex base-editing (MBE) platform that significantly improves the capability and throughput of fungal genome manipulation, leading to the simultaneous inactivation of up to eight genes using a single transformation. We then employed MBE to inactivate three negative epigenetic regulators combinatorially in Aspergillus nidulans, enabling the activation of eight cryptic gene clusters compared to the wild-type strains. A group of novel NPs harboring unique cichorine and polyamine hybrid chemical scaffolds were identified, which were not reported previously. We envision that our scalable and efficient MBE platform can be readily applied in other filamentous fungi for the genome mining of novel NPs, providing a powerful approach for the exploitation of fungal chemical diversity.

Structural basis of the stereoselective formation of the spirooxindole ring in the biosynthesis of citrinadins.

Prenylated indole alkaloids featuring spirooxindole rings possess a 3R or 3S carbon stereocenter, which determines the bioactivities of these compounds. Despite the stereoselective advantages of spirooxindole biosynthesis compared with those of organic synthesis, the biocatalytic mechanism for controlling the 3R or 3S-spirooxindole formation has been elusive. Here, we report an oxygenase/semipinacolase CtdE that specifies the 3S-spirooxindole construction in the biosynthesis of 21R-citrinadin A. High-resolution X-ray crystal structures of CtdE with the substrate and cofactor, together with site-directed mutagenesis and computational studies, illustrate the catalytic mechanisms for the possible ß-face epoxidation followed by a regioselective collapse of the epoxide intermediate, which triggers semipinacol rearrangement to form the 3S-spirooxindole. Comparing CtdE with PhqK, which catalyzes the formation of the 3R-spirooxindole, we reveal an evolutionary branch of CtdE in specific 3S spirocyclization. Our study provides deeper insights into the stereoselective catalytic machinery, which is important for the biocatalysis design to synthesize spirooxindole pharmaceuticals.

Quinolactacin Biosynthesis Involves Non-Ribosomal-Peptide-Synthetase-Catalyzed Dieckmann Condensation to Form the Quinolone-γ-lactam Hybrid.

Quinolactacins are novel fungal alkaloids that feature a quinolone-γ-lactam hybrid, which is a potential pharmacophore for the treatment of cancer and Alzheimer's disease. Herein, we report the identification of the quinolactacin A2 biosynthetic gene cluster and elucidate the enzymatic basis for the formation of the quinolone-γ-lactam structure. We reveal an unusual ß-keto acid (N-methyl-2-aminobenzoylacetate) precursor that is derived from the primary metabolite l-kynurenine via methylation, oxidative decarboxylation, and amide hydrolysis reactions. In vitro assays reveal two single-module non-ribosomal peptide synthetases (NRPs) that incorporate the ß-keto acid and l-isoleucine, followed by Dieckmann condensation, to form the quinolone-γ-lactam. Notably, the bioconversion from l-kynurenine to the ß-keto acid is a unique strategy employed by nature to decouple R*-domain-containing NRPS from the polyketide synthase (PKS) machinery, expanding the paradigm for the biosynthesis of quinolone-γ-lactam natural products via Dieckmann condensation.

Promotion of compound K production in Saccharomyces cerevisiae by glycerol.

BACKGROUND: Ginsenoside compound K (CK), one of the primary active metabolites of protopanaxadiol-type ginsenosides, is produced by the intestinal flora that degrade ginseng saponins and exhibits diverse biological properties such as anticancer, anti-inflammatory, and anti-allergic properties. However, it is less abundant in plants. Therefore, enabling its commercialization by construction of a Saccharomyces cerevisiae cell factory is of considerable significance. RESULTS: We induced overexpression of PGM2, UGP1, and UGT1 genes in WLT-MVA5, and obtained a strain that produces ginsenoside CK. The production of CK at 96 h was 263.94 ± 2.36 mg/L, and the conversion rate from protopanaxadiol (PPD) to ginsenoside CK was 64.23 ± 0.41%. Additionally, it was observed that the addition of glycerol was beneficial to the synthesis of CK. When 20% glucose (C mol) in the YPD medium was replaced by the same C mol glycerol, CK production increased to 384.52 ± 15.23 mg/L, which was 45.68% higher than that in YPD medium, and the PPD conversion rate increased to 77.37 ± 3.37% as well. As we previously observed that ethanol is beneficial to the production of PPD, ethanol and glycerol were fed simultaneously in the 5-L bioreactor fed fermentation, and the CK levels reached 1.70 ± 0.16 g/L. CONCLUSIONS: In this study, we constructed an S. cerevisiae cell factory that efficiently produced ginsenoside CK. Glycerol effectively increased the glycosylation efficiency of PPD to ginsenoside CK, guiding higher carbon flow to the synthesis of ginsenosides and effectively improving CK production. CK production attained in a 5-L bioreactor was 1.7 g/L after simultaneous feeding of glycerol and ethanol.

High-titer production of 13R-manoyl oxide in metabolically engineered Saccharomyces cerevisiae.

BACKGROUND: Diterpenoids are a large class of natural products with complex structures and broad commercial applications as food additives, important medicines, and fragrances. However, their low abundance in plants and high structural complexity limit their applications. Therefore, it is important to create an efficient diterpenoid-producing yeast cell factory of the production of various high-value diterpenoid compounds in a cost-effective manner RESULTS: In this study, 13R-manoyl oxide (13R-MO; 2.31 mg/L) was produced by expressing CfTPS2 and CfTPS3 from Coleus forskohlii in Saccharomyces cerevisiae. The 13R-MO titer was increased by 142-fold to 328.15 mg/L via the stepwise metabolic engineering of the original strain, including the overexpression of the rate-limiting genes (tHMG1 and ERG20) of the mevalonate pathway, transcription and protein level regulation of ERG9, Bts1p and Erg20F96Cp fusion, and the overexpression of tCfTPS2 and tCfTPS3 (excision of the N-terminal plastid transit peptide sequences of CfTPS2 and CfTPS3). The final titer of 13R-MO reached up to 3 g/L by fed-batch fermentation in a 5 L bioreactor. CONCLUSIONS: In this study, an efficient 13R-MO yeast cell factory was constructed, which achieved the de novo production of 3 g/L of 13R-MO from glucose. To the best of our knowledge, this is the highest 13R-MO titer reported to date. Furthermore, the metabolic engineering strategies presented here could be used to produce other valuable diterpenoid compounds in yeast.

Engineering Saccharomyces cerevisiae for Enhanced Production of Protopanaxadiol with Cofermentation of Glucose and Xylose.

Protopanaxadiol (PPD), an active triterpene compound, is the precursor of high-value ginsenosides. In this study, we report a strategy for the enhancement of PPD production in Saccharomyces cerevisiae by cofermentation of glucose and xylose. In mixed sugar fermentation, strain GW6 showed higher PPD titer and yield than that obtained from glucose cultivation. Then, engineering strategies were implemented on GW6 to enhance the PPD yields, such as adjustment of the central carbon metabolism, optimization of the mevalonate pathway, reinforcement of the xylose assimilation pathway, and regulation of cofactor balance, namely, overexpression of xPK/PTA, ERG10/ERG12/ERG13, XYL1/XYL2/TAL1, and POS5, respectively. In particular, the final obtained strain GW10, harboring overexpressed POS5, exhibited the highest PPD yield, which was 2.06 mg of PPD/g of mixed sugar. In a 5-L fermenter, PPD titer reached 152.37 mg/L. These promising results demonstrate the great advantages of mixed sugar over glucose for high-yield production of PPD.

Production of sesquiterpenoid zerumbone from metabolic engineered Saccharomyces cerevisiae.

Zerumbone, the predominant sesquiterpenoid component of Zingiber zerumbet, exhibits diverse pharmacological properties. In this study, de novo production of zerumbone was achieved in a metabolically engineered yeast cell factory by introducing α-humulene synthase (ZSS1), α-humulene 8-hydroxylase (CYP71BA1) and zerumbone synthase variant (ZSD1S114A) from Z. zerumbet, together with AtCPR1 from Arabidopsis thaliana into the yeast strain. Multistep metabolic engineering strategies were applied, including the over-expression of the mevalonate (MVA) pathway rate-limiting enzymes tHMG1 and ERG20, regulation of ERG9 by an inducible promoter and competitive pathway deletion to redirect metabolic flux toward the desired product. In the engineered strain, α-humulene production increased by 18-fold, to 92 mg/L compared to that in the original strain. Five cytochrome P450 reductases (CPRs) from different sources were selected for CYP71BA1 adaptability tests, and AtCPR1 from A. thaliana was found to be the optimal, producing 113.16 µg/L of 8-hydroxy-α-humulene. Multicopy integration of CYP71BA1, AtCPR1, ZSS1 and ICE2 (type III membrane protein) genes resulting in strain LW14 increased the production of 8-hydroxy-α-humulene by 134-fold to 15.2 mg/L. Expressing ZSD1S114A in the ura3 site of strain LW14 resulted in the production of 7 mg/L zerumbone. Multicopy integration of ZSD1S114A increased the production of zerumbone to 20.6 mg/L. The high zerumbone-producing strain was used for batch and fed-batch fermentation in a 5-L bioreactor and zerumbone degradation by yeast was observed; the production of zerumbone finally reached 40 mg/L by fed-batch fermentation in a 5-L bioreactor.

Semicontinuous sophorolipid fermentation using a novel bioreactor with dual ventilation pipes and dual sieve-plates coupled with a novel separation system.

Sophorolipids (SLs) are biosurfactants with widespread applications. The yield and purity of SLs are two important factors to be considered during their commercial large-scale production. Notably, SL accumulation causes an increase in viscosity, decrease in dissolved oxygen and product inhibition in the fermentation medium. This inhibits the further production and purification of SLs. This describes the development of a novel integrated system for SL production using Candida albicans O-13-1. Semicontinuous fermentation was performed using a novel bioreactor with dual ventilation pipes and dual sieve-plates (DVDSB). SLs were separated and recovered using a newly designed two-stage separation system. After SL recovery, the fermentation broth containing residual glucose and oleic acid was recycled back into the bioreactor. This novel approach considerably alleviated the problem of product inhibition and accelerated the rate of substrate utilization. Production of SLs achieved was 477 g l-1 , while their productivity was 1.59 g l-1  h-1 . Purity of SLs improved by 23.3%, from 60% to 74%, using DVDSB with the separation system. The conversion rate of carbon source increased from 0.5 g g-1 (in the batch fermentation) to 0.6 g g-1 . These results indicated that the integrated system could improve the efficiency of production and purity of SLs.

Heterologous biosynthesis of triterpenoid ambrein in engineered Escherichia coli.

OBJECTIVES: To genetically engineer Escherichia coli for the heterologous biosynthesis of triterpenoid, ambrein, the main bioactive component of ambergris, by constituting a novel squalene-derived ambrein biosynthetic pathway in E. coli. RESULTS: The ScERG9 gene encoding the squalene synthase (SS) was integrated into the E. coli genome to generate a squalene-producing strain that supplied the central precursor squalene for the formation of cyclic triterpenoids. The mutated squalene-hopene synthase (D377C SHC) and the tetraprenyl-ß-curcumene cyclase (BmeTC) were co-expressed with SS to construct a novel ambrein biosynthetic pathway in E. coli. Ambrein was produced at 2.6 mg l-1. CONCLUSIONS: An E. coli chassis for ambrein production was constructed by combining the squalene synthesis module with the downstream cyclization module.

Metabolomics analysis of the effect of dissolved oxygen on spinosad production by Saccharopolyspora spinosa.

Spinosad, a universal bio-pesticide, is obtained from the soil actinomycete Saccharopolyspora spinosa. Dissolved oxygen, an important contributing factor in aerobic microbial fermentation, however, is not always available in sufficient amounts. To alleviate oxygen limitation in spinosad production, three different oxygen vectors, namely oleic acid, toluene, and n-dodecane, were added into early fermentation. Results indicated that n-dodecane was the optimal oxygen vector. Spinosad yield was increased by 44.2% compared to that in the control group in the presence of 0.5% n-dodecane, added after 120 h of incubation. Yields of the test group reached 6.52 mg/g dry cell weight (DCW), while that of the control group was limited to 4.52 mg/g DCW. Metabolomics analysis by gas chromatography coupled to mass spectrometry was performed to demonstrate the metabolism mechanism in the presence and absence of oxygen vector. In total, 78 principal intracellular metabolites in S. spinosa were detected and quantified in the presence and absence of n-dodecane. Levels of some metabolites that were related to the tricarboxylic acid cycle and pentose phosphate pathway varied significantly. Aspartic acid and glucose-1-phosphate levels varied significantly and contributed most in the distinction of the fermentation conditions and phases. The above findings give new insights into the improvement and the metabolomic characteristics of industrial spinosad production.

Enhancing Saccharomyces cerevisiae reactive oxygen species and ethanol stress tolerance for high-level production of protopanoxadiol.

Protopanaxadiol (PPD) is an active compound in Panax ginseng. Recently, an optimized PPD synthesis pathway contained a ROS releasing step (a P450-type PPD synthase, PPDS) was introduced into Saccharomyces cerevisiae. Here reported a synergistic effect of PPDS-CPR (CPR, cytochrome P450 reductase) uncoupling and ethanol stress on ROS releasing, which reduced cells viability. To build a robust strain, a cell wall integrity associated gene SSD1 was high-expressed to improve ethanol tolerance, and ROS level decreased for 24.7%. Then, regulating the expression of an oxidative stress regulation gene YBP1 decreased 75.2% of ROS releasing, and improved cells viability from 71.3±1.3% to 88.3±1.4% at 84h. Increased cells viability enables yeast to produce more PPD through feeding additional ethanol. In 5L fermenter, PPD production of W3a-ssPy reached to 4.25±0.18g/L (19.48±0.28mg/L/OD600), which is the highest yield reported so far. This work makes the industrial production of PPD possible by microbial fermentation.

Optimization of a cytochrome P450 oxidation system for enhancing protopanaxadiol production in Saccharomyces cerevisiae.

Ginsenosides, the major bioactive components of Panax ginseng, are regarded as promising high-value pharmaceutical compounds. In ginseng, ginsenosides are produced from their precursor protopanaxadiol. Recently, an artificial biosynthetic pathway of protopanaxadiol was built in Saccharomyces cerevisiae by introducing a P. ginseng dammarenediol-II synthase, a P. ginseng cytochrome P450-type protopanaxadiol synthase (PPDS), and a Arabidopsis thaliana NADPH-cytochrome P450 reductase (ATR1). In this engineered yeast strain, however, the low metabolic flux through PPDS resulted in a low productivity of protopanaxadiol. Moreover, health of the yeast cells was significantly affected by reactive oxygen species released by the pool coupling between PPDS and ATR1. To overcome the obstacles in protopanaxadiol production, PPDS was modified through transmembrane domain truncation and self-sufficient PPDS-ATR1 fusion construction in this study. The fusion enzymes conferred approximately 4.5-fold increase in catalytic activity, and 71.1% increase in protopanaxadiol production compared with PPDS and ATR1 co-expression. Our in vivo experiment indicated that the engineered yeast carrying fusion protein effectively converted 96.8% of dammarenediol-II into protopanaxadiol. Protopanaxadiol production in a 5 L bioreactor in fed-batch fermentation reached 1436.6 mg/L. Our study not only improved protopanaxadiol production in yeast, but also provided a generic method to improve activities of plant cytochrome P450 monooxygenases. This method is promising to be applied to other P450 systems in yeast. Biotechnol. Bioeng. 2016;113: 1787-1795. © 2016 Wiley Periodicals, Inc.

Heterologous biosynthesis of triterpenoid dammarenediol-II in engineered Escherichia coli.

OBJECTIVES: To achieve heterologous biosynthesis of dammarenediol-II, which is the precursor of dammarane-type tetracyclic ginsenosides, by reconstituting the 2,3-oxidosqualene-derived triterpenoid biosynthetic pathway in Escherichia coli. RESULTS: By the strategy of synthetic biology, dammarenediol-II biosynthetic pathway was reconstituted in E. coli by co-expression of squalene synthase (SS), squalene epoxidase (SE), NADPH-cytochrome P450 reductase (CPR) from Saccharomyces cerevisiae, and SE from Methylococcus capsulatus (McSE), NADPH-cytochrome P450 reductase (CPR) from Arabidopsis thaliana. Sequences of transmembrane domains were truncated if necessary in each of the genes. Different sources of SE/CPR combinations were tested, during which two CPRs were detected to be new reductase partners of McSE. When the gene encoding dammarenediol-II synthase was co-expressed with the 2,3-oxidosqualene expression modules, dammarenediol-II was detected and the production was 8.63 mg l(-1) in E. coli under the shake-flask conditions. CONCLUSIONS: Two E. coli chassis for production of dammarenediol-II were established which could be potentially applied in other triterpenoid production in E. coli when different oxidosqualene cyclases (OSCs) introduced into the system.

Coupling of Spinosad Fermentation and Separation Process via Two-Step Macroporous Resin Adsorption Method.

In this paper, a two-step resin adsorption technology was investigated for spinosad production and separation as follows: the first step resin addition into the fermentor at early cultivation period to decrease the timely product concentration in the broth; the second step of resin addition was used after fermentation to adsorb and extract the spinosad. Based on this, a two-step macroporous resin adsorption-membrane separation process for spinosad fermentation, separation, and purification was established. Spinosad concentration in 5-L fermentor increased by 14.45 % after adding 50 g/L macroporous at the beginning of fermentation. The established two-step macroporous resin adsorption-membrane separation process got the 95.43 % purity and 87 % yield for spinosad, which were both higher than that of the conventional crystallization of spinosad from aqueous phase that were 93.23 and 79.15 % separately. The two-step macroporous resin adsorption method has not only carried out the coupling of spinosad fermentation and separation but also increased spinosad productivity. In addition, the two-step macroporous resin adsorption-membrane separation process performs better in spinosad yield and purity.

Suitable extracellular oxidoreduction potential inhibit rex regulation and effect central carbon and energy metabolism in Saccharopolyspora spinosa.

BACKGROUND: Polyketides, such as spinosad, are mainly synthesized in the stationary phase of the fermentation. The synthesis of these compounds requires many primary metabolites, such as acetyl-CoA, propinyl-CoA, NADPH, and succinyl-CoA. Their synthesis is also significantly influenced by NADH/NAD+. Rex is the sensor of NADH/NAD+ redox state, whose structure is under the control of NADH/NAD+ ratio. The structure of rex controls the expression of many NADH dehydrogenases genes and cytochrome bd genes. Intracellular redox state can be influenced by adding extracellular electron acceptor H2O2. The effect of extracellular oxidoreduction potential on spinosad production has not been studied. Although extracellular oxidoreduction potential is an important environment effect in polyketides production, it has always been overlooked. Thus, it is important to study the effect of extracellular oxidoreduction potential on Saccharopolyspora spinosa growth and spinosad production. RESULTS: During stationary phase, S. spinosa was cultured under oxidative (H2O2) and reductive (dithiothreitol) conditions. The results show that the yield of spinosad and pseudoaglycone increased 3.11 fold under oxidative condition. As H2O2 can be served as extracellular electron acceptor, the ratios of NADH/NAD+ were measured. We found that the ratio of NADH/NAD+ under oxidative condition was much lower than that in the control group. The expression of cytA and cytB in the rex mutant indicated that the expression of these two genes was controlled by rex, and it was not activated under oxidative condition. Enzyme activities of PFK, ICDH, and G6PDH and metabolites results indicated that more metabolic flux flow through spinosad synthesis. CONCLUSION: The regulation function of rex was inhibited by adding extracellular electron acceptor-H2O2 in the stationary phase. Under this condition, many NADH dehydrogenases which were used to balance NADH/NAD+ by converting useful metabolites to useless metabolites and unefficient terminal oxidases (cytochrome bd) were not expressed. So lots of metabolites were not waste to balance. As a result, un-wasted metabolites related to spinosad and PSA synthesis resulted in a high production of spinosad and PSA under oxidative condition.

A comparative metabolomics analysis of Saccharopolyspora spinosa WT, WH124, and LU104 revealed metabolic mechanisms correlated with increases in spinosad yield.

Metabolomics analysis of three Saccharopolyspora spinosa strains (wild type strain WT, ultraviolet mutant strain WH124, and metabolic engineering strain LU104) with different spinosad producing levels was performed by liquid chromatograph coupled to mass spectrometry (LC-MS). The metabolite profiles were subjected to hierarchal clustering analysis (HCA) and principal component analysis (PCA). The results of HCA on a heat map revealed that the large numbers of primary metabolism detected were more abundant in WH124 and less abundant in LU104 during the early fermentation stage as compared to the WT strain. PCA separated the three strains clearly and suggested nine metabolites that contributed predominantly to the separation. These biomarkers were associated with central carbon metabolism (succinic acid, α-ketoglutarate, acetyl-CoA, and ATP), amino acid metabolism (glutamate, glutamine, and valine), and secondary metabolism (pseudoaglycone), etc. These findings provide insight into the metabolomic characteristics of the two high-yield strains and for further regulation of spinosad production.