Synthetic biology advances towards a bio-based society in the era of artificial intelligence.

Synthetic biology is a rapidly emerging field with broad underlying applications in health, industry, agriculture, or environment, enabling sustainable solutions for unmet needs of modern society. With the very recent addition of artificial intelligence (AI) approaches, this field is now growing at a rate that can help reach the envisioned goals of bio-based society within the next few decades. Integrating AI with plant-based technologies, such as protein engineering, phytochemicals production, plant system engineering, or microbiome engineering, potentially disruptive applications have already been reported. These include enzymatic synthesis of new-to-nature molecules, bioelectricity generation, or biomass applications as construction material. Thus, in the not-so-distant future, synthetic biologists will help attain the overarching goal of a sustainable yet efficient production system for every aspect of society.

Enhanced production of withaferin A from the hairy root culture of Withania somnifera via synergistic effect of Methyl jasmonate and ß-cyclodextrin.

Due to low amounts of withanolides produced in some plants and high demand for various applications, their biotechnological production is widely researched. The effects of two explant types (i.e., leaf and stem from the in vitro seedlings of three genotypes of Withania somnifera) and four Rhizobium strains (i.e., LBA 9402, A4, ATCC 15834, and C58C1) to improve hairy root formation efficiency was studied. Furthermore, the combined effects of ß-cyclodextrin (ß-CD) and methyl jasmonate (MeJA) on withaferin A production after 48 h exposure time was examined. Four hairy roots having the maximum percentage of induced roots and mean number of induced roots to analyze their growth kinetics and identified G3/ATCC/LEAF culture having the maximum specific growth rate (µ = 0.036 day-1) and growth index (GI = 9.18), and the shortest doubling time (Td = 18.82 day) were selected. After 48 h exposure of G3/ATCC/LEAF culture to different elicitation conditions, maximum amounts of withaferin A were produced in samples co-treated with 0.5 mM ß-CD + 100 µM MeJA (9.57 mg/g DW) and 5.0 mM ß-CD + 100 µM MeJA (17.45 mg/g DW). These outcomes represented a 6.8-fold and 12.5-fold increase, respectively, compared to the control. Similarly, combined ß-CD/MeJA elicitation increased gene expression levels of HMGR, SQS, SMT-1, and SDS/CYP710A involved in withanolides biosynthetic pathway, of which just SMT-1 had significant correlation with withaferin A production. These results demonstrated the superiority of G1-leaf explant and ATCC 15834 for hairy root induction, and revealed synergistic effect of MeJA and ß-CD on withaferin A production.

Widespread biosynthesis of 16-carbon terpenoids in bacteria.

Terpenoids are the most diverse group of specialized metabolites with numerous applications. Their biosynthesis is based on the five-carbon isoprene building block and, as a result, almost all terpenoids isolated to date are based on backbones that contain multiples of five carbon atoms. Intrigued by the discovery of an unusual bacterial terpenoid with a 16-carbon skeleton, here we investigate whether the biosynthesis of 16-carbon terpenoids is more widespread than this single example. We mine bacterial genomic information and identify potential C16 biosynthetic clusters in more than 700 sequenced genomes. We study selected clusters using a yeast synthetic biology platform and reveal that the encoded synthases produce at least 47 different noncanonical terpenoids. By thorough chemical analysis, we explain the structures of 13 C16 metabolites, most of which possess intricate highly strained bi- and tricyclic backbones. Our results unveil the existence of an extensive class of terpenoids in bacteria.

Engineering yeast for the production of plant terpenoids using synthetic biology approaches.

Covering: 2011-2022The low amounts of terpenoids produced in plants and the difficulty in synthesizing these complex structures have stimulated the production of terpenoid compounds in microbial hosts by metabolic engineering and synthetic biology approaches. Advances in engineering yeast for terpenoid production will be covered in this review focusing on four directions: (1) manipulation of host metabolism, (2) rewiring and reconstructing metabolic pathways, (3) engineering the catalytic activity, substrate selectivity and product specificity of biosynthetic enzymes, and (4) localizing terpenoid production via enzymatic fusions and scaffolds, or subcellular compartmentalization.

Engineering membrane architecture for biotechnological applications.

Cellular membranes, predominantly described as a dynamic bilayer, are composed of different lipids, transmembrane proteins, and carbohydrates. Most research on biological membranes focuses on the identification, characterization, and mechanistic aspects of their different components. These studies provide a fundamental understanding of membrane structure, function, and dynamics, establishing a basis for the development of membrane engineering strategies. To date, approaches in this field concentrate on membrane adaptation to harsh conditions during industrial fermentation, which can be caused by temperature, osmotic, or organic solvent stress. With advances in the field of metabolic engineering and synthetic biology, recent breakthroughs include proof of concept microbial production of essential medicines, such as cannabinoids and vinblastine. However, long pathways, low yields, and host adaptation continue to pose challenges to the efficient scale up production of many important compounds. The lipid bilayer is profoundly linked to the activity of heterologous membrane-bound enzymes and transport of metabolites. Therefore, strategies for improving enzyme performance, facilitating pathway reconstruction, and enabling storage of products to increase the yields directly involve cellular membranes. At the forefront of membrane engineering research are re-emerging approaches in lipid research and synthetic biology that manipulate membrane size and composition and target lipid profiles across species. This review summarizes engineering strategies applied to cellular membranes and discusses the challenges and future perspectives, particularly with regards to their applications in host engineering and bioproduction.

Expanding the terpene biosynthetic code with non-canonical 16 carbon atom building blocks.

Humankind relies on specialized metabolites for medicines, flavors, fragrances, and numerous other valuable biomaterials. However, the chemical space occupied by specialized metabolites, and, thus, their application potential, is limited because their biosynthesis is based on only a handful of building blocks. Engineering organisms to synthesize alternative building blocks will bypass this limitation and enable the sustainable production of molecules with non-canonical chemical structures, expanding the possible applications. Herein, we focus on isoprenoids and combine synthetic biology with protein engineering to construct yeast cells that synthesize 10 non-canonical isoprenoid building blocks with 16 carbon atoms. We identify suitable terpene synthases to convert these building blocks into C16 scaffolds and a cytochrome P450 to decorate the terpene scaffolds and produce different oxygenated compounds. Thus, we reconstruct the modular structure of terpene biosynthesis on 16-carbon backbones, synthesizing 28 different non-canonical terpenes, some of which have interesting odorant properties.

Transforming yeast peroxisomes into microfactories for the efficient production of high-value isoprenoids.

Current approaches for the production of high-value compounds in microorganisms mostly use the cytosol as a general reaction vessel. However, competing pathways and metabolic cross-talk frequently prevent efficient synthesis of target compounds in the cytosol. Eukaryotic cells control the complexity of their metabolism by harnessing organelles to insulate biochemical pathways. Inspired by this concept, herein we transform yeast peroxisomes into microfactories for geranyl diphosphate-derived compounds, focusing on monoterpenoids, monoterpene indole alkaloids, and cannabinoids. We introduce a complete mevalonate pathway in the peroxisome to convert acetyl-CoA to several commercially important monoterpenes and achieve up to 125-fold increase over cytosolic production. Furthermore, peroxisomal production improves subsequent decoration by cytochrome P450s, supporting efficient conversion of (S)-(-)-limonene to the menthol precursor trans-isopiperitenol. We also establish synthesis of 8-hydroxygeraniol, the precursor of monoterpene indole alkaloids, and cannabigerolic acid, the cannabinoid precursor. Our findings establish peroxisomal engineering as an efficient strategy for the production of isoprenoids.

Integrating pathway elucidation with yeast engineering to produce polpunonic acid the precursor of the anti-obesity agent celastrol.

BACKGROUND: Celastrol is a promising anti-obesity agent that acts as a sensitizer of the protein hormone leptin. Despite its potent activity, a sustainable source of celastrol and celastrol derivatives for further pharmacological studies is lacking. RESULTS: To elucidate the celastrol biosynthetic pathway and reconstruct it in Saccharomyces cerevisiae, we mined a root-transcriptome of Tripterygium wilfordii and identified four oxidosqualene cyclases and 49 cytochrome P450s as candidates to be involved in the early steps of celastrol biosynthesis. Using functional screening of the candidate genes in Nicotiana benthamiana, TwOSC4 was characterized as a novel oxidosqualene cyclase that produces friedelin, the presumed triterpenoid backbone of celastrol. In addition, three P450s (CYP712K1, CYP712K2, and CYP712K3) that act downstream of TwOSC4 were found to effectively oxidize friedelin and form the likely celastrol biosynthesis intermediates 29-hydroxy-friedelin and polpunonic acid. To facilitate production of friedelin, the yeast strain AM254 was constructed by deleting UBC7, which afforded a fivefold increase in friedelin titer. This platform was further expanded with CYP712K1 to produce polpunonic acid and a method for the facile extraction of products from the yeast culture medium, resulting in polpunonic acid titers of 1.4 mg/L. CONCLUSION: Our study elucidates the early steps of celastrol biosynthesis and paves the way for future biotechnological production of this pharmacologically promising compound in engineered yeast strains.

Orthogonal monoterpenoid biosynthesis in yeast constructed on an isomeric substrate.

Synthetic biology efforts for the production of valuable chemicals are frequently hindered by the structure and regulation of the native metabolic pathways of the chassis. This is particularly evident in the case of monoterpenoid production in Saccharomyces cerevisiae, where the canonical terpene precursor geranyl diphosphate is tightly coupled to the biosynthesis of isoprenoid compounds essential for yeast viability. Here, we establish a synthetic orthogonal monoterpenoid pathway based on an alternative precursor, neryl diphosphate. We identify structural determinants of isomeric substrate selectivity in monoterpene synthases and engineer five different enzymes to accept the alternative substrate with improved efficiency and specificity. We combine the engineered enzymes with dynamic regulation of metabolic flux to harness the potential of the orthogonal substrate and improve the production of industrially-relevant monoterpenes by several-fold compared to the canonical pathway. This approach highlights the introduction of synthetic metabolism as an effective strategy for high-value compound production.

Isoprenoid biosynthesis in the diatom Haslea ostrearia.

Diatoms are eukaryotic, unicellular algae that are responsible for c. 20% of the Earth's primary production. Their dominance and success in contemporary oceans have prompted investigations on their distinctive metabolism and physiology. One metabolic pathway that remains largely unexplored in diatoms is isoprenoid biosynthesis, which is responsible for the production of numerous molecules with unique features. We selected the diatom species Haslea ostrearia because of its characteristic isoprenoid content and carried out a comprehensive transcriptomic analysis and functional characterization of the genes identified. We functionally characterized one farnesyl diphosphate synthase, two geranylgeranyl diphosphate synthases, one short-chain polyprenyl synthase, one bifunctional isopentenyl diphosphate isomerase - squalene synthase, and one phytoene synthase. We inferred the phylogenetic origin of these genes and used a combination of functional analysis and subcellular localization predictions to propose their physiological roles. Our results provide insight into isoprenoid biosynthesis in H. ostrearia and propose a model of the central steps of the pathway. This model will facilitate the study of metabolic pathways of important isoprenoids in diatoms, including carotenoids, sterols and highly branched isoprenoids.

Synthesis of 11-carbon terpenoids in yeast using protein and metabolic engineering.

One application of synthetic biology is the redesign of existing biological systems to acquire new functions. In this context, expanding the chemical code underlying key biosynthetic pathways will lead to the synthesis of compounds with new structures and potentially new biological activities. Terpenoids are a large group of specialized metabolites with numerous applications. Yet, being synthesized from five-carbon units, they are restricted to distinct classes that differ by five carbon atoms (C10, C15, C20, etc.). To expand the diversity of terpenoid structures, we engineered yeast cells to synthesize a noncanonical building block with 11 carbons, and produced 40 C11 terpene scaffolds that can form the basis for an entire terpenoid class. By identifying a single-residue switch that converts C10 plant monoterpene synthases to C11-specific enzymes, we engineered dedicated synthases for C11 terpene production. This approach will enable the systematic expansion of the chemical space accessed by terpenoids.

Overcoming the plasticity of plant specialized metabolism for selective diterpene production in yeast.

Plants synthesize numerous specialized metabolites (also termed natural products) to mediate dynamic interactions with their surroundings. The complexity of plant specialized metabolism is the result of an inherent biosynthetic plasticity rooted in the substrate and product promiscuity of the enzymes involved. The pathway of carnosic acid-related diterpenes in rosemary and sage involves promiscuous cytochrome P450s whose combined activity results in a multitude of structurally related compounds. Some of these minor products, such as pisiferic acid and salviol, have established bioactivity, but their limited availability prevents further evaluation. Reconstructing carnosic acid biosynthesis in yeast achieved significant titers of the main compound but could not specifically yield the minor products. Specific production of pisiferic acid and salviol was achieved by restricting the promiscuity of a key enzyme, CYP76AH24, through a single-residue substitution (F112L). Coupled with additional metabolic engineering interventions, overall improvements of 24 and 14-fold for pisiferic acid and salviol, respectively, were obtained. These results provide an example of how synthetic biology can help navigating the complex landscape of plant natural product biosynthesis to achieve heterologous production of useful minor metabolites. In the context of plant adaptation, these findings also suggest a molecular basis for the rapid evolution of terpene biosynthetic pathways.

Carnosic acid biosynthesis elucidated by a synthetic biology platform.

Synthetic biology approaches achieving the reconstruction of specific plant natural product biosynthetic pathways in dedicated microbial "chassis" have provided access to important industrial compounds (e.g., artemisinin, resveratrol, vanillin). However, the potential of such production systems to facilitate elucidation of plant biosynthetic pathways has been underexplored. Here we report on the application of a modular terpene production platform in the characterization of the biosynthetic pathway leading to the potent antioxidant carnosic acid and related diterpenes in Salvia pomifera and Rosmarinus officinalis.Four cytochrome P450 enzymes are identified (CYP76AH24, CYP71BE52, CYP76AK6, and CYP76AK8), the combined activities of which account for all of the oxidation events leading to the biosynthesis of the major diterpenes produced in these plants. This approach develops yeast as an efficient tool to harness the biotechnological potential of the numerous sequencing datasets that are increasingly becoming available through transcriptomic or genomic studies.

Production of the forskolin precursor 11ß-hydroxy-manoyl oxide in yeast using surrogate enzymatic activities.

BACKGROUND: Several plant diterpenes have important biological properties. Among them, forskolin is a complex labdane-type diterpene whose biological activity stems from its ability to activate adenylyl cyclase and to elevate intracellular cAMP levels. As such, it is used in the control of blood pressure, in the protection from congestive heart failure, and in weight-loss supplements. Chemical synthesis of forskolin is challenging, and production of forskolin in engineered microbes could provide a sustainable source. To this end, we set out to establish a platform for the production of forskolin and related epoxy-labdanes in yeast. RESULTS: Since the forskolin biosynthetic pathway has only been partially elucidated, and enzymes involved in terpene biosynthesis frequently exhibit relaxed substrate specificity, we explored the possibility of reconstructing missing steps of this pathway employing surrogate enzymes. Using CYP76AH24, a Salvia pomifera cytochrome P450 responsible for the oxidation of C-12 and C-11 of the abietane skeleton en route to carnosic acid, we were able to produce the forskolin precursor 11ß-hydroxy-manoyl oxide in yeast. To improve 11ß-hydroxy-manoyl oxide production, we undertook a chassis engineering effort involving the combination of three heterozygous yeast gene deletions (mct1/MCT1, whi2/WHI2, gdh1/GDH1) and obtained a 9.5-fold increase in 11ß-hydroxy-manoyl oxide titers, reaching 21.2 mg L(-1). CONCLUSIONS: In this study, we identify a surrogate enzyme for the specific and efficient hydroxylation of manoyl oxide at position C-11ß and establish a platform that will facilitate the synthesis of a broad range of tricyclic (8,13)-epoxy-labdanes in yeast. This platform forms a basis for the heterologous production of forskolin and will facilitate the elucidation of subsequent steps of forskolin biosynthesis. In addition, this study highlights the usefulness of using surrogate enzymes for the production of intermediates of complex biosynthetic pathways. The combination of heterozygous deletions and the improved yeast strain reported here will provide a useful tool for the production of numerous other isoprenoids.

Combined metabolome and transcriptome profiling provides new insights into diterpene biosynthesis in S. pomifera glandular trichomes.

BACKGROUND: Salvia diterpenes have been found to have health promoting properties. Among them, carnosic acid and carnosol, tanshinones and sclareol are well known for their cardiovascular, antitumor, antiinflammatory and antioxidant activities. However, many of these compounds are not available at a constant supply and developing biotechnological methods for their production could provide a sustainable alternative. The transcriptome of S.pomifera glandular trichomes was analysed aiming to identify genes that could be used in the engineering of synthetic microbial systems. RESULTS: In the present study, a thorough metabolite analysis of S. pomifera leaves led to the isolation and structure elucidation of carnosic acid-family metabolites including one new natural product. These labdane diterpenes seem to be synthesized through miltiradiene and ferruginol. Transcriptomic analysis of the glandular trichomes from the S. pomifera leaves revealed two genes likely involved in miltiradiene synthesis. Their products were identified and the corresponding enzymes were characterized as copalyl diphosphate synthase (SpCDS) and miltiradiene synthase (SpMilS). In addition, several CYP-encoding transcripts were identified providing a valuable resource for the identification of the biosynthetic mechanism responsible for the production of carnosic acid-family metabolites in S. pomifera. CONCLUSIONS: Our work has uncovered the key enzymes involved in miltiradiene biosynthesis in S. pomifera leaf glandular trichomes. The transcriptomic dataset obtained provides a valuable tool for the identification of the CYPs involved in the synthesis of carnosic acid-family metabolites.

Use of the de novo transcriptome analysis of silver-leaf nightshade (Solanum elaeagnifolium) to identify gene expression changes associated with wounding and terpene biosynthesis.

BACKGROUND: Solanum elaeagnifolium, an invasive weed of the Solanaceae family, is poorly studied although it poses a significant threat to crops. Here the analysis of the transcriptome of S. elaeagnifolium is presented, as a means to explore the biology of this species and to identify genes related to its adaptation to environmental stress. One of the basic mechanisms by which plants respond to environmental stress is through the synthesis of specific secondary metabolites that protect the plant from herbivores and microorganisms, or serve as signaling molecules. One important such group of secondary metabolites are terpenes. RESULTS: By next-generation sequencing, the flower/leaf transcriptome of S. elaeagnifolium was sequenced and de novo assembled into 75,618 unigenes. Among the unigenes identified, several corresponded to genes involved in terpene biosynthesis; these included terpene synthases (TPSs) and genes of the mevalonate (MVA) and the methylerythritol phosphate (MEP) pathways. Functional characterization of two of the TPSs showed that one produced the sesquiterpene (E)-caryophyllene and the second produced the monoterpene camphene. Analysis of wounded S. elaeagnifolium leaves has shown significant increase of the concentration of (E)-caryophyllene and geranyl linalool, two terpenes implicated in stress responses. The increased production of (E)-caryophyllene was matched to the induced expression of the corresponding TPS gene. Wounding also led to the increased expression of the putative 1-deoxy-D-xylulose-5-phosphate synthase 2 (DXS2) gene, a key enzyme of the MEP pathway, corroborating the overall increased output of terpene biosynthesis. CONCLUSIONS: The reported S. elaeagnifolium de novo transcriptome provides a valuable sequence database that could facilitate study of this invasive weed and contribute to our understanding of the highly diverse Solanaceae family. Analysis of genes and pathways involved in the plant's interaction with the environment will help to elucidate the mechanisms that underly the intricate features of this unique Solanum species.

Iterative carotenogenic screens identify combinations of yeast gene deletions that enhance sclareol production.

BACKGROUND: Terpenoids (isoprenoids) have numerous applications in flavors, fragrances, drugs and biofuels. The number of microbially produced terpenoids is increasing as new biosynthetic pathways are being elucidated. However, efforts to improve terpenoid production in yeast have mostly taken advantage of existing knowledge of the sterol biosynthetic pathway, while many additional factors may affect the output of the engineered system. RESULTS: Aiming to develop a yeast strain that can support high titers of sclareol, a diterpene of great importance for the perfume industry, we sought to identify gene deletions that improved carotenoid, and thus potentially sclareol, production. Using a carotenogenic screen, the best 100 deletion mutants, out of 4,700 mutant strains, were selected to create a subset for further analysis. To identify combinations of deletions that cooperate to further boost production, iterative carotenogenic screens were applied, and each time the top performing gene deletions were further ranked according to the number of genetic and physical interactions known for each specific gene. The gene selected in each round was deleted and the resulting strain was employed in a new round of selection. This approach led to the development of an EG60 derived haploid strain combining six deletions (rox1, dos2, yer134c, vba5, ynr063w and ygr259c) and exhibiting a 40-fold increase in carotenoid and 12-fold increase in sclareol titers, reaching 750 mg/L sclareol in shake flask cultivation. CONCLUSION: Using an iterative approach, we identified novel combinations of yeast gene deletions that improve carotenoid and sclareol production titers without compromising strain growth and viability. Most of the identified deletions have not previously been implicated in sterol pathway control. Applying the same approach using a different starting point could yield alternative sets of deletions with similar or improved outcome.

Efficient diterpene production in yeast by engineering Erg20p into a geranylgeranyl diphosphate synthase.

Terpenes have numerous applications, ranging from pharmaceuticals to fragrances and biofuels. With increasing interest in producing terpenes sustainably and economically, there has been significant progress in recent years in developing methods for their production in microorganisms. In Saccharomyces cerevisiae, production of the 20-carbon diterpenes has so far proven to be significantly less efficient than production of their 15-carbon sesquiterpene counterparts. In this report, we identify the modular structure of geranylgeranyl diphosphate synthesis in yeast to be a major limitation in diterpene yields, and we engineer the yeast farnesyl diphosphate synthase Erg20p to produce geranylgeranyl diphosphate. Using a combination of protein and genetic engineering, we achieve significant improvements in the production of sclareol and several other isoprenoids, including cis-abienol, abietadiene and ß-carotene. We also report the development of yeast strains carrying the engineered Erg20p, which support efficient isoprenoid production and can be used as a dedicated chassis for diterpene production or biosynthetic pathway elucidation. The design developed here can be applied to the production of any GGPP-derived isoprenoid and is compatible with other yeast terpene production platforms.

Reconstructing the chemical diversity of labdane-type diterpene biosynthesis in yeast.

Terpenes are a large class of natural products, many of which are used in cosmetics, pharmaceuticals, or biofuels. However, terpene's industrial application is frequently hindered by limited availability of natural sources or low yields of chemical synthesis. In this report, we developed a modular platform based on standardized and exchangeable parts to reproduce and potentially expand the diversity of terpene structures in Saccharomyces cerevisiae. By combining different module-specific parts, we exploited the substrate promiscuity of class I diterpene synthases to produce an array of labdane-type scaffolds. These were subsequently modified by a scaffold decoration module consisting of a mutant library of a promiscuous cytochrome P450 to afford a range of hydroxylated diterpenes. Further P450 protein engineering yielded dedicated and efficient catalysts for specific products. Terpenes produced include precursors of pharmacologically important compounds, molecules that are difficult to obtain from natural sources, or new natural products. The approach described here provides a platform on which additional gene mining, combinatorial biosynthesis, and protein engineering efforts can be integrated to sustainably explore the terpene chemical space.

Engineering monoterpene production in yeast using a synthetic dominant negative geranyl diphosphate synthase.

Monoterpenes have an established use in the food and cosmetic industries and have recently also found application as advanced biofuels. Although metabolic engineering efforts have so far achieved significant yields of larger terpenes, monoterpene productivity is lagging behind. Here, we set out to establish a monoterpene-specific production platform in Saccharomyces cerevisiae and identified the sequential reaction mechanism of the yeast farnesyl diphosphate synthase Erg20p to be an important factor limiting monoterpene yield. To overcome this hurdle, we engineered Erg20p into a geranyl diphosphate synthase and achieved a significant increase in monoterpene titers. To further improve production, we converted the engineered geranyl diphosphate synthase into a dominant negative form, so as to decrease the ability of the endogenous Erg20p to function as a farnesyl diphosphate synthase, without entirely abolishing sterol biosynthesis. Fusion of the synthetic dominant negative Erg20p variant with the terpene synthase, combined with yeast strain engineering, further improved monoterpene yields and achieved an overall 340-fold increase in sabinene yield over the starting strain. The design described here can be readily incorporated to any dedicated yeast strain, while the developed plasmid vectors and heterozygous ERG20 deletion yeast strain can also be used as a plug-and-play system for enzyme characterization and monoterpene pathway elucidation.