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Plant natural products (PNPs) exhibit a wide range of biological activities and have essential applications in various fields such as medicine, agriculture, and flavors. Given their natural limitations, the production of high-value PNPs using microbial cell factories has become an effective alternative in recent years. However, host metabolic burden caused by its massive accumulation has become one of the main challenges for efficient PNP production. Therefore, it is necessary to strengthen the transmembrane transport process of PNPs. This review introduces the discovery and mining of PNP transporters to directly mediate PNP transmembrane transportation both intracellularly and extracellularly. In addition to transporter engineering, this review also summarizes several auxiliary strategies (such as small molecules, environmental changes, and vesicles assisted transport) for strengthening PNP transportation. Finally, this review is concluded with the applications and future perspectives of transportation engineering in the construction and optimization of PNP microbial cell factories.

Abstract While sanguinarine has gained recognition for antimicrobial and antineoplastic activities, its complex conjugated structure and low abundance in plants impede broad applications. Here, we demonstrate the complete biosynthesis of sanguinarine and halogenated derivatives using highly engineered yeast strains. To overcome sanguinarine cytotoxicity, we establish a splicing intein-mediated temperature-responsive gene expression system (SIMTeGES), a simple strategy that decouples cell growth from product synthesis without sacrificing protein activity. To debottleneck sanguinarine biosynthesis, we identify two reticuline oxidases and facilitated functional expression of flavoproteins and cytochrome P450 enzymes via protein molecular engineering. After comprehensive metabolic engineering, we report the production of sanguinarine at a titer of 448.64 mg L−1. Additionally, our engineered strain enables the biosynthesis of fluorinated sanguinarine, showcasing the biotransformation of halogenated derivatives through more than 15 biocatalytic steps. This work serves as a blueprint for utilizing yeast as a scalable platform for biomanufacturing diverse benzylisoquinoline alkaloids and derivatives.

Abstract The antiarrhythmic drug ajmaline is a monoterpenoid indole alkaloid (MIA) isolated from the Ayurvedic plant Rauvolfia serpentina (Indian Snakeroot). Research into the biosynthesis of ajmaline and another renowned MIA chemotherapeutic drug vinblastine has yielded pivotal advancements in the fields of plant specialized metabolism and engineering over recent decades. While the majority of vinblastine biosynthesis has been recently elucidated, the quest for comprehending ajmaline biosynthesis remains incomplete, marked by the absence of two critical enzymes. Here, we show the discovery and characterization of these two elusive reductases, alongside the identification of two physiologically relevant esterases that complete the biosynthesis of ajmaline. We show that ajmaline biosynthesis proceeds with vomilenine 1,2(R)-reduction followed by its 19,20(S)-reduction. This process is further modulated by two root-expressing esterases that deacetylate 17-O-acetylnorajmaline. Expanding upon the successful completion of the ajmaline biosynthetic pathway, we engineer the de novo biosynthesis of ajmaline in Baker’s yeast.

γ-Linolenic acid-enriched galactosyldiacylglycerols (GDGs-GLA), as the natural form of γ-linolenic acid in microalgae, have a range of functional activities, including anti-inflammatory, antioxidant, and anti-allergic properties. The low abundance of microalgae and the structural stereoselectivity complexity impede microalgae extraction or chemical synthesis, resulting in a lack of supply of GDGs-GLA with a growing demand. At present, there is a growing interest in engineering oleaginous yeasts for mass production of GDGs-GLA based on their ability to utilize a variety of hydrophobic substrates and a high metabolic flux toward fatty acid and lipid (triacylglycerol, TAG) production. Here, we first introduce the GDGs-GLA biosynthetic pathway in microalgae and challenges in the engineering of the native host. Subsequently, we describe in detail the applications of oleaginous yeasts with Yarrowia lipolytica as the representative for GDGs-GLA biosynthesis, including the development of synthetic biology parts, gene editing tools, and metabolic engineering of lipid biosynthesis. Finally, we discuss the development trend of GDGs-GLA biosynthesis in Y. lipolytica.

Abstract β-Elemene, an active ingredient found in medicinal plants like turmeric and zedoary, is a sesquiterpene compound with antitumor activity against various cancers. However, its current mode of production through plant extraction suffers from low efficiency and limited natural resources. Recently, there has been an increased interest in establishing microbial cell factories to produce germacrene A, which can be converted to β-elemene by a one-step reaction in vitro. In this study, we constructed an engineered Pichia pastoris cell factory for producing germacrene A. We rerouted the fluxes towards germacrene A biosynthesis through the optimization of the linker sequences between germacrene A synthase (GAS) and farnesyl pyrophosphate synthase (ERG20), overexpression of important pathway genes (i.e., IDI1, tHMG1, and ACS), and multi-copy integration of related expression cassettes. In combination with medium optimization and bioprocess engineering, the final titer of germacrene A in a 1 L fermenter reached 1.9 g/L through fed-batch fermentation. This represents the first report on the production of germacrene A in P. pastoris and demonstrates its advantage in producing terpenoids and other value-added natural products. Graphical Abstract

Abstract Esters are widely used in food, energy, spices, chemical industry, etc., becoming an indispensable part of life. However, their production heavily relies on the fossil energy industry, which presents significant challenges associated with energy shortages and environmental pollution. Consequently, there is an urgent need to identify alternative green methods for ester production. One promising solution is biosynthesis, which offers sustainable and environmentally friendly processes. In ester biosynthesis, alcohol acyltransferases (AATs) catalyze the condensation of acyl-CoAs and alcohols to form esters, enabling the biosynthesis of nearly 100 different kinds of esters, such as ethyl acetate, hexyl acetate, ethyl crotonate, isoamyl acetate, and butyl butyrate. However, low catalytic efficiency and low selectivity of AATs represent the major bottlenecks for the biosynthesis of certain specific esters, which should be addressed with protein molecular engineering approaches before practical biotechnological applications. This review provides an overview of AAT enzymes, including their sequences, structures, active sites, catalytic mechanisms, and metabolic engineering applications. Furthermore, considering the critical role of AATs in determining the final ester products, the current research progresses of AAT modification using protein molecular engineering are also discussed. This review summarized the major challenges and prospects of AAT enzymes in ester biosynthesis.

Paclitaxel is a renowned broad-spectrum anticancer drug. With the establishment of a chromosome-level high-quality reference genome map of Taxus, recent research on paclitaxel biosynthesis has flourished. The oxetane ring is a distinctive chemical moiety of paclitaxel, and three recent studies have proposed different enzymes involved in its formation, reflecting divergent opinions on whether the pathway proceeds via acetylation followed by epoxidation or vice versa. Subsequently, researchers have elucidated gene clusters responsible for the biosynthesis of the key intermediate baccatin III. Despite varying reports, two studies successfully achieved heterologous biosynthesis of baccatin III by transient expression in tobacco. Taxadiene 5α-hydroxylase (T5αH), the first cytochrome P450 in the pathway, exhibited varied product profiles upon heterologous expression systems, contrasting with observations in native Taxus species, probably due to differences in partner proteins or cellular microenvironments. Further elucidation of biosynthesis mechanisms, including the reaction order and the promiscuity of key enzymes, is anticipated through collaborative efforts among botanists, chemists, and synthetic biologists.

Brassinolide (BL) is one of the most active compounds among phytohormone brassinosteroids (BRs) and can be used for plant growth and development regulation. Here, the authors report the construction of an artificial pathway in baker’s yeast for scalable production of 24-epi-ergosterol, a precursor for BL semi-synthesis.

Abstract Background Ralstonia solanacearum, one of the most devastating bacterial plant pathogens, is the causal agent of bacterial wilt. Recently, several studies on resistance to bacterial wilt have been conducted using the Arabidopsis-R. solanacearum system. However, the progress of R. solanacearum infection in Arabidopsis is still unclear. Results We generated a bioluminescent R. solanacearum by expressing plasmid-based luxCDABE. Expression of luxCDABE did not alter the bacterial growth and pathogenicity. The light intensity of bioluminescent R. solanacearum was linearly related to bacterial concentrations from 104 to 108 CFU·mL−1. After root inoculation with bioluminescent R. solanacearum strain, light signals in tomato and Arabidopsis were found to be transported from roots to stems via the vasculature. Quantification of light intensity from the bioluminescent strain accurately reported the difference in disease resistance between Arabidopsis wild type and resistant mutants. Conclusions Bioluminescent R. solanacearum strain spatially and quantitatively measured bacterial growth in tomato and Arabidopsis, and offered a tool for the high-throughput study of R. solanacearum-Arabidopsis interaction in the future.

Modulating the extracellular matrix microenvironment is critical for achieving the desired macrophage phenotype in immune investigations or tumor therapy. Combining de novo protein design and biosynthesis techniques, herein, we designed a biomimetic polypeptide self-assembled nano-immunomodulator to trigger the activation of a specific macrophage phenotype. It was intended to be made up of (​GGS​GGP​GGG​PAS​AAA​NSA​SRA​TSN​SP)n, the RGD motif from collagen, and the IKVAV motif from laminin. The combination of these domains allows the biomimetic polypeptide to assemble into extracellular matrix-like nanofibrils, creating an extracellular matrix-like milieu for macrophages. Furthermore, changing the concentration further provides a facile route to fine-tune macrophage polarization, which enhances antitumor immune responses by precisely resetting tumor-associated macrophage immune responses into an M1-like phenotype, which is generally considered to be tumor-killing macrophages, primarily antitumor, and immune-promoting. Unlike metal or synthetic polymer-based nanoparticles, this polypeptide-based nanomaterial exhibits excellent biocompatibility, high efficacy, and precise tunability in immunomodulatory effectiveness. These encouraging findings motivate us to continue our research into cancer immunotherapy applications in the future.

Abstract Background CO2 valorization is one of the effective methods to solve current environmental and energy problems, in which microbial electrosynthesis (MES) system has proved feasible and efficient. Cupriviadus necator (Ralstonia eutropha) H16, a model chemolithoautotroph, is a microbe of choice for CO2 conversion, especially with the ability to be employed in MES due to the presence of genes encoding [NiFe]-hydrogenases and all the Calvin–Benson–Basham cycle enzymes. The CO2 valorization strategy will make sense because the required hydrogen can be produced from renewable electricity independently of fossil fuels. Main body In this review, synthetic biology toolkit for C. necator H16, including genetic engineering vectors, heterologous gene expression elements, platform strain and genome engineering, and transformation strategies, is firstly summarized. Then, the review discusses how to apply these tools to make C. necator H16 an efficient cell factory for converting CO2 to value-added products, with the examples of alcohols, fatty acids, and terpenoids. The review is concluded with the limitation of current genetic tools and perspectives on the development of more efficient and convenient methods as well as the extensive applications of C. necator H16. Conclusions Great progress has been made on genetic engineering toolkit and synthetic biology applications of C. necator H16. Nevertheless, more efforts are expected in the near future to engineer C. necator H16 as efficient cell factories for the conversion of CO2 to value-added products.

Liu et al. optimize vindoline biosynthesis in yeast from tabersonine by altering the copy numbers of pathway genes, pairing CYPs with CPRs, engineering the microenvironment for functional expression of CYPs, enhancing cofactor supplies, and optimizing fermentation conditions. By doing so, the authors increased the production of vindoline to a final titer as high as ∼16.5 mg/L.

The methylotrophic yeast Pichia pastoris (a.k.a. Komagataella phaffii) is one of the most commonly used hosts for industrial production of recombinant proteins. As a non-conventional yeast, P. pastoris has unique biological characteristics and its expression system has been well developed. With the advances in synthetic biology, more efforts have been devoted to developing P. pastoris into a chassis for the production of various high-value compounds, such as natural products. This review begins with the introduction of synthetic biology tools for the engineering of P. pastoris, including vectors, promoters, and terminators for heterologous gene expression as well as Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated System (CRISPR/Cas) for genome editing. This review is then followed by examples of the production of value-added natural products in metabolically engineered P. pastoris strains. Finally, challenges and outlooks in developing P. pastoris as a synthetic biology chassis are prospected.

Triacetic acid lactone (TAL) is a promising renewable platform polyketide with broad biotechnological applications. In this study, we constructed an engineered Pichia pastoris strain for the production of TAL. We first introduced a heterologous TAL biosynthetic pathway by integrating the 2-pyrone synthase encoding gene from Gerbera hybrida (Gh2PS). We then removed the rate-limiting step of TAL synthesis by introducing the posttranslational regulation-free acetyl-CoA carboxylase mutant encoding gene from S. cerevisiae (ScACC1*) and increasing the copy number of Gh2PS. Finally, to enhance intracellular acetyl-CoA supply, we focused on the introduction of the phosphoketolase/phosphotransacetylase pathway (PK pathway). To direct more carbon flux towards the PK pathway for acetyl-CoA generation, we combined it with a heterologous xylose utilization pathway or endogenous methanol utilization pathway. The combination of the PK pathway with the xylose utilization pathway resulted in the production of 825.6 mg/L TAL in minimal medium with xylose as the sole carbon source, with a TAL yield of 0.041 g/g xylose. This is the first report on TAL biosynthesis in P. pastoris and its direct synthesis from methanol. The present study suggests potential applications in improving the intracellular pool of acetyl-CoA and provides a basis for the construction of efficient cell factories for the production of acetyl-CoA derived compounds.

Vinblastine has been used clinically as one of the most potent therapeutics for the treatment of several types of cancer. However, the traditional plant extraction method suffers from unreliable supply, low abundance, and extremely high cost. Here, we use synthetic biology approach to engineer Saccharomyces cerevisiae for de novo biosynthesis of vindoline and catharanthine, which can be coupled chemically or biologically to vinblastine. On the basis of a platform strain with sufficient supply of precursors and cofactors for biosynthesis, we reconstituted, debottlenecked, and optimized the biosynthetic pathways for the production of vindoline and catharanthine. The vindoline biosynthetic pathway represents one of the most complicated pathways ever reconstituted in microbial cell factories. Using shake flask fermentation, our engineered yeast strains were able to produce catharanthine and vindoline at a titer of 527.1 and 305.1 μg·liter−1, respectively, without accumulating detectable amount of pathway intermediates. This study establishes a representative example for the production of valuable plant natural products in yeast.

Genome-scale engineering is generally limited to single methods of alteration such as overexpression, repression or deletion. Here the authors present a tri-functional CRISPR system that can engineer complex synergistic interactions in a genome-wide manner.

Cytochrome P450 enzymes (P450s) are a superfamily of heme-thiolate proteins widely existing in various organisms and play a key role in the metabolic network and secondary metabolism. However, the low expression levels and activities have become the biggest challenge for P450s studies. To improve the functional expression of P450s in Saccharomyces cerevisiae, an Arabidopsis thaliana cDNA library was expressed in the betaxanthin-producing yeast strain, which functioned as a biosensor for high throughput screening. Three new target genes AtGRP7, AtMSBP1, and AtCOL4 were identified to improve the functional expression of CYP76AD1 in yeast, with accordingly the accumulation of betaxanthin increased for 1.32-, 1.86-, and 1.10-fold, respectively. In addition, these three targets worked synergistically/additively to improve the production of betaxanthin, representing a total of 2.36-fold improvement when compared with the parent strain. More importantly, these genes were also determined to effectively increase the activity of another P450 enzyme (CYP736A167), catalyzing the hydroxylation of α-santalene to produce Z-α-santalol. Simultaneous overexpression of AtGRP7, AtMSBP1, and AtCOL4 increased α-santalene to Z-α-santalol conversion rate for more than 2.97-fold. The present study reported a novel strategy to improve the functional expression of P450s in S. cerevisiae and promises the construction of platform yeast strains for the production of natural products.

Existing efforts have been focused on one of the elements in the automation of the design, build, test, and learn (DBTL) cycle for biosystems design. Here, the authors integrate a robotic system with machine learning algorithms to fully automate the DBTL cycle and apply it in optimizing the lycopene biosynthetic pathway.

The introduction of multi-gene metabolic pathways is generally the first step for the construction of microbial cell factories and plays an essential role in metabolic engineering and synthetic biology. Here, we developed a “PCR & Go” system for facile integration and assembly of multi-gene pathways into the chromosome of Saccharomyces cerevisiae. The core component of the “PCR & Go” system was an expression chassis, where eight promoter/terminator pairs were pre-installed into the yeast chromosome and PCR amplified gene fragments could be inserted directly for functional expression. In combination with the CRISPR/Cas9 system and a gRNA plasmid library, the β-carotene (three genes), zeaxanthin (four genes), and astaxanthin (five genes) biosynthetic pathways were integrated and assembled into the yeast genome with an efficiency of ~93, ~85, and 69%, respectively, using PCR amplified gene fragments with ~40 bp homology arms in a single step. Therefore, the “PCR & Go” system can be used for fast construction of yeast cell factories harboring multi-gene pathways with high efficiency and flexibility.

Crocetin is a plant natural product with broad medicinal applications, such as improvement of sleep quality and attenuation of physical fatigue. However, crocetin production using microbial cell factories is still far from satisfaction, probably due to the conflict between cell growth and product accumulation. In the present work, a temperature-responsive crocetin-producing Saccharomyces cerevisiae strain was established to coordinate cell growth, precursor (zeaxanthin) generation, and product (crocetin) biosynthesis. The production of crocetin was further enhanced via increasing the copy numbers of CCD2 and ALDH genes using the CRISPR-Cas9 based multiplex genome integration technology. The final engineered strain TL009 produced crocetin up to 139.67 ± 2.24 μg/g DCW. The advantage of the temperature switch based crocetin production was particularly demonstrated by much higher zeaxanthin conversion yield. This study highlights the potential of the temperature-responsive yeast platform strains to increase the production of other valuable carotenoid derivatives.

Advances in metabolic engineering and synthetic biology have facilitated the manufacturing of many valuable-added compounds and commodity chemicals using microbial cell factories in the past decade. However, due to complexity of cellular metabolism, the optimization of metabolic pathways for maximal production represents a grand challenge and an unavoidable barrier for metabolic engineering. Recently, cell-free protein synthesis system (CFPS) has been emerging as an enabling alternative to address challenges in biomanufacturing. This review summarizes the recent progresses of CFPS in rapid prototyping of biosynthetic pathways and genetic circuits (biosensors) to speed up design-build-test (DBT) cycles of metabolic engineering and synthetic biology. Keywords: Cell-free protein synthesis, Metabolic pathway optimization, Genetic circuits, Metabolic engineering, Synthetic biology

Metaboli engineering through gene overexpression, knock-down and knock-out is often carried out sequentially in a high labor, low-throughput manner. Here, the authors use CRISPR-mediated gene activation, interference and deletion to rapidly rewire S. cerevisiae metabolism in a single step.

The continuous progress in the structural and functional characterization of aquaporins increasingly attracts attention to study their roles in certain mammalian diseases. Although several structures of aquaporins have already been solved by crystallization, the challenge of producing sufficient amounts of functional proteins still remains. CF (cell free) expression has emerged in recent times as a promising alternative option in order to synthesize large quantities of membrane proteins, and the focus of this report was to evaluate the potential of this technique for the production of eukaryotic aquaporins. We have selected the mouse aquaporin 4 as a representative of mammalian aquaporins. The protein was synthesized in an E. coli extract based cell-free system with two different expression modes, and the efficiencies of two modes were compared. In both, the P-CF (cell-free membrane protein expression as precipitate) mode generating initial aquaporin precipitates as well as in the D-CF (cell-free membrane protein expression in presence of detergent) mode, generating directly detergent solubilized samples, we were able to obtain mg amounts of protein per ml of cell-free reaction. Purified aquaporin samples solubilized in different detergents were reconstituted into liposomes, and analyzed for the water channel activity. The calculated P(f) value of proteoliposome samples isolated from the D-CF mode was 133 µm/s at 10°C, which was 5 times higher as that of the control. A reversible inhibitory effect of mercury chloride was observed, which is consistent with previous observations of in vitro reconstituted aquaporin 4. In this study, a fast and convenient protocol was established for functional expression of aquaporins, which could serve as basis for further applications such as water filtration.