Microbial synthetic biology for plant metabolite production: a strategy to reconcile human health with the realization of the UN Sustainable Development Goals

Plants produce a wide range of secondary metabolites that provide an array of benefits for human health. Challenges of native host production systems such as excessive land use, large carbon footprints, long production times, and high production costs often make plant‐based production of these critical metabolites inefficient and unsustainable. Engineered production through microbial‐based synthetic biology offers an alternative method of producing plant compounds that addresses the limitations encountered in plant‐based production. New technological and analytical tools such as bioprospecting, machine learning, and protein modeling can be used to explore biodiversity‐based data to identify more efficient enzymatic sequences for optimizing the production of plant derivatives. Ex planta production using microbial chassis systems provides a flexible, scalable metabolic platform that can more readily integrate new metabolic processes. Potential improvements include increased production rates and enhanced bioavailability of critical compounds, expanding the frontiers of metabolite production for optimized clinical applications. Microbial‐based synthetic biology also opens an avenue for sustainable production, with the capability of modifying the microbial chassis to accommodate a wide variety of substrates for feedstock. It includes the utilization of waste from human activities as carbon sources for production, presenting an opportunity to use cheap renewable resources for greener production methods. The benefits of ex planta production systems can potentially enable the efficient, scalable, and sustainable production of plant derivatives that may vastly improve health and help bring forward the realization of the 2030 UN Sustainable Development Goals.


I
t is widely recognized that the earth's ecosystems are being modified in an unprecedented way due to human activity.The United Nations has established a series of Sustainable Development Goals (SDGs) in 2015 to manage these impacts, seeking to improve the wellbeing of people and the planet alike by 2030. 1 In that regard, new sustainable production systems need to be implemented to cover the current and future demands of our growing population, and to ease the environmental burden experienced by global ecosystems.
Synthetic biology (SynBio), at its core, combines and incorporates genetic material (genes, promoters, transcription factors) from different organisms into an expression chassis (mainly microorganisms but superior organisms have also been explored) to obtain a desired output.Examples of engineered microbial outputs are plant-derived bioproducts with therapeutical and pharmaceutical applications that overcome the complications encountered with native host systems in plant-based production.Metabolic engineering is a key component of the microbial-based production, where it combines the biological building blocks provided by SynBio to create a suitable production pathway. 2 A growing number of tools are available for the comprehension, optimization, and expression of metabolic paths; 3 a prime example is clustered regularly interspaced short palindromic repeats or (CRISPR) for adding, activating, or inhibiting targeted genes. 4ioprospecting biodiversity serves as a rich source of 'building blocks' for synthesizing biomolecules (enzymes), as well as for the discovery of novel chassis. 5In this regard, advances in next-generation sequencing technologies have contributed substantially to the amount of data available from all seven kingdoms, creating an extraordinary database for SynBio applications. 6Combining these technologies, it is possible to recover full biosynthetic pathways for de novo reconstruction of plant metabolites of interest from sequencing data.
Microbial SynBio has emerged as a portion of synthetic biology that employs microorganisms to produce high-value bioproducts.The support that SynBio may offer on the way to sustainable goals was clearly discussed in the comment article reported by K.E.French. 7In this article, we discuss our perspective on how microbial SynBio can pave the way towards sustainable production of valuable plant metabolites for improving human health, accomplishing SDG 3, 'Good Health and Well-being' .A sustainable production system should be implemented in a way where land use is minimized, valuable ecosystem functions are protected, and consequently biodiversity (almost an unlimited source of information) is not lost.In our view, it is essential to generate production systems for a circular economy, where waste residues from human activities can be exploited as substrates.

Plants versus microbial cell factories
To date, homologous systems (native host) are the main source of plant metabolites but this plant-based production system has several limitations.The extensive use of arable land, expensive inputs, and high demand for water resources make plant-based production methods unsustainable.The transformation of wild landscapes into agricultural fields are a major driver of biodiversity loss.Current plant-breeding programs focus mainly on production traits (drought, flood, and disease tolerance) that may lead to a decay in the natural selection of micronutrients and general secondary metabolite levels. 8Additionally, the production time needed to achieve enough biomass to recover the target compound is relatively long compared with microbial production and is also dependent on seasonal weather.Most plant natural products are expressed in low quantities and are tissue-specific, so bioproduct production is often difficult.As an example, some ginsenosides for the plant Panax ginseng are present almost as traces within a mix of different compounds, making the extraction and purification processes inefficient. 9Finally, many secondary metabolites are overproduced only under biotic and abiotic stress conditions, with elicitors needed to enhance the plant-based production of plant derivatives.For instance, the overexpression of genes IFS1 and IFS2 and consequently accumulation of isoflavones in soybean is only increased in the presence of elicitors such as Aspergillus niger, jasmonic acid, and salicylic acid, which contribute to production costs. 10Complex downstream processes, especially during extraction and purification, also add to production costs.An example is the production of curcumin, the main active component of Curcuma longa (turmeric).Enzyme-assisted, supercritical CO 2 , microwave-assisted, and green solvent extractions each improve curcumin production rates but the average yield of less than 5% make these approaches both time demanding and cost ineffective. 11he application of microbial SynBio can provide an avenue to overcome the limitations previously described.Growing microorganisms with the ability to produce plant bioproducts 1487 is a scalable, landless form of production that is not reliant on seasonal weather and can potentially utilize inputs/feedstock that are cost effective, along with simpler and less problematic purification processes.Different organisms are used to produce plant derivatives, with the broadly recognized chassis Escherichia coli and Saccharomyces cerevisiae being the most cited.Escherichia coli and S. cerevisiae are model species with vast availability of supporting genetic engineering toolkits, and their culturing conditions and scalability have already been optimized. 12Escherichia coli has a faster duplication rate, and it naturally produces a set of metabolites that are different to those produced by plants, so the heterologous expression can be easily identified.However, as plant metabolite production usually requires part of the eukaryotic transmembrane proteins such as P450, S. cerevisiae is the best candidate.Prokaryotic organisms can be provided with plant machinery but this approach reduces the growth rate and increases the metabolic burden for long enzymatic pathways. 13Furthermore, eukaryotic organisms contain most of the subcellular compartments found in plants, which makes them more suitable hosts when the production of intermediate molecules needs to be compartmentalized.As S. cerevisiae is a fungal species that is generally recognized as safe (GRAS), it is a good candidate for safe heterologous expression. 14Less well known microorganisms are utilized as microbial factories for producing natural products.We recommend the review by Pham et al. for more details regarding this field of study. 15

Microbial SynBio for human health and wellbeing
There are extensive examples in which microbial SynBio was used to produce plant metabolites such as flavonoids, alkaloids, betalains, and glucosinolates. 16A representation of the production approach is presented in Fig. 1.Considering the advantages, microbial-based production appears to be a better strategy than homologous production but several aspects need to be assessed.The biosynthetic pathway for a candidate drug first needs to be identified and, if it has been well characterized, then it is a straightforward process to transfer the genes into the specific microbial host.As an example, resveratrol, a recognized antioxidant, was successfully produced using E. coli as a production system. 17n the other hand, if the biosynthesis pathway is unknown, further strategies are compulsory, such as exploring a comprehensive transcriptome from cultivars that express the target compound for the optimal metabolic candidate.From there, genes with different expression levels can be targeted, and enzymatic activities can be tested in-silico using protein docking. 18This approach was used to detect Perspective: Microbial synthetic biology for plant metabolite production candidate genes for coumestrol, a plant metabolite from soybean with multiple health benefits. 19Another strategy for recreating the full metabolic pathway uses retro-analysis to retro-build the pathway, identifying precursor compounds from the desired metabolite. 20Moreover, taking advantage of all the omics data already generated, is it now possible to use machine-and deep-learning strategies to build pathways and model metabolomics trees for different species. 21nce the potential pathway is defined, bioprospecting techniques and combinatorial biosynthesis can be used to find more efficient enzymes from different organisms, which can be linked together to build a biosynthetic route with higher productivity. 5That was the case for curcuminoids (anti-inflammatory and antioxidant properties), where a combinatorial strategy was used to obtain 1529.5 μM of curcumin in E. Coli. 22o overcome the metabolic burden resulting from the incorporation of additional genes into one strain, a co-culture strategy can be followed where the metabolic pathway of interest is separated and integrated into different strains. 23ne extreme example is the implementation of polycultures, where complex molecules such as anthocyanins (another kind of plant derivate with a variety of medicinal applications, apart from being colored water-soluble pigments) were produced from simple sugars using a mix of several strains of E. coli. 24his approach can also generate a greater diversification of pathways and products, and open options for future developments.In cases where the whole pathway has yet to be identified or when the pathway includes several enzymatic steps, a combined strategy can be performed using chemical biosynthesis and microbial SynBio.As an example, high-yield production of artemisin, a plant derivative used for treating malaria, is achieved by feeding the culture with the precursor amorpha-4,11-diene, obtaining 40 g/L of artemisinin acid.Artemisinin acid is then converted into artemisin using a simple chemical synthesis step. 25One concern for producing human health derivatives is the excessive use of antibiotics during the production phase.To solve this issue, E. coli strains were engineered to be auxotrophic, using amino acids supplementation instead of antibiotic as a selection marker. 26n summary, microbial SynBio can be used to produce plant derivatives and it offers boundless alternatives for exploiting the power of microbes to produce bioproducts that support human health and well-being.

Sustainable production systems
From our perspective, to fulfill the 2030 UN SDGs, circular bio-economies utilizing new organisms and substrate types need to be tested as alternative solutions.
This bioeconomic approach reduces production costs but also assists in diminishing the environmental footprint through the development of carbon-negative production systems.Approaches for achieving this goal include, (a) using model organisms acting as chassis that can be engineered to process both agro-industrial wastes or C1/C2 (CO 2 , methane, ethanol) molecules, or (b) the discovery of novel non-model microorganisms that use those sources naturally, both of which are key contenders to explore for the sustainable production of desirable compounds. 27A list of examples involving microbial production systems are provided below and summarized in Table 1.

Lignin as source of carbon and precursors
One solution is to use model organisms with pre-treated waste residues as precursors for feedstock, with lignin as an example for coumarin production in S. cerevisiae.When lignin is broken down chemically with an alkaline solution, one of the main monomers obtained is ferulic acid.Ferulic acid is a precursor, used for the production of scopoletin, a coumarin used for treating rheumatic arthritis. 28A further important consideration for optimizing the production of value-added products from lignin is the mitigation of inhibitors from lignocellulosic biomass that can reduce the production yield. 29Several strategies were explored to identify novel microorganisms and molecular pathways for the ability to degrade lignin without the need for any pre-treatment to diversify production methods for future bio-manufacturing strategies. 30

Using methanotrophic yeast
The yeast Pichia pastoris is widely used as a heterologous system, such as those seen in S. cerevisiae, but the supporting engineering tools are limited in comparison and it is used primarily for recombinant protein production.There are some examples of P. pastoris strains used for producing metabolites from methanol.However, these efforts were focused on fungi bioproducts, such as lovastatin and monacolin J, rather than for plant metabolites. 31The use of P. pastoris shows growing potential, with a strain that was successfully engineered to fix CO 2 expanding the metabolic frontiers of this organism. 32

Agro-industrial waste residues as feedstock
The bacterial genus Streptomyces are valuable bioprospecting targets due to its highly diverse metabolic plasticity to produce a plethora of bioactive metabolites.This genus also has the ability to use agricultural and forestry plant waste as a carbon source. 34Currently, there are no reports of plant derivatives produced using this microbe, but studies have examined expansion of the taxa's biosynthetic capabilities beyond bacterial compounds.An example is the strain Streptomyces fulvissimus CKS7, which shows ability to produce bioethanol using agricultural waste as a carbon source. 35Other waste products from human activity can be used as feedstock.For instance, the thermophile microbe Parageobacillus thermoglucosidasius was engineered for terpene production (plant metabolite with multiple clinical applications) using waste bread as a carbon source. 36

CO 2 sequestrating bacteria
Another approach is to engineer model microorganisms that use CO 2 as a carbon source, as seen in Third Generation Microbial Biorefineries.An autotroph converted E. coli strain was tested at laboratory scale for producing biomass from CO 2 , although no production of plant natural products was achieved so far using this strategy. 37Non-model microbes can be also used for producing plant metabolites from CO 2 .Cyanobacteria, a prokaryotic microalgae with the ability to naturally express P450 proteins needed for plant metabolite production, has demonstrated the ability to produce plant compounds such as carotenoids. 38A drawback is the cost of transportation and accumulation of CO 2 , which has prevented the use of non-model microbes from being competitive against other carbon sources.Consequently, many of the well-established companies that use thirdgeneration Biorefineries are focusing on commodities instead of specific pharmaceuticals, limiting development in healthrelated bioproduct production using this approach. 39croalgae double impact: CO 2 sequestration and wastewater treatment Microalgae are photosynthetic unicellular organisms that naturally use CO 2 and light to create biomass, which can be utilized to produce microbial-based bioproducts such as bioethanol, as well as bioactive food and medicinal products.40 Wastewater can also be used as a source of nitrogen, phosphorus, and other micronutrients for microalgae-based microbial production, with the additional potential to serve as a as water treatment system for improving water quality.Microalgae photo-bioreactors are also scalable, with the potential capacity to create proteinlaced food products for coping with growing demand, and also for the obtention of microalgae-derived compounds with beneficial applications for human health.41 As microalgae and land plants share evolutionary ancestors, this potential compatibility can be further explored to produce plant natural products.Species such as Chlorophyta reinhardtii and Phaeodactylum tricornutum are already engineered for the production of isoprenoids, one of the largest groups of plant-derived metabolites used for clinical purposes.42 Some microalgae species such as Chlorella vulgaris can also be cocultured with S. cerevisiae, creating a symbiotic relationship interchanging carbon sources and precursors, allowing the metabolic burden to be shared, and improving the efficiency of metabolite production.43

Better bioavailability
Microbial SynBio has the potential to provide sustainable production of a wide range of well-known plant metabolites but it can also be used to improve the pharmacokinetics properties of these compounds.One of the main variables for increasing therapeutic efficacy is bioavailability -if the compounds are rapidly degraded or are unable to reach their physiological targets, the bioproduct in question may not be suitable for use in clinical applications.Microbial SynBio can increase bioavailability through molecular modifications and expand product branches for future discoveries, diversifying the enzymatic steps within the metabolic pathway.Promiscuous enzymes that accept different substrates as input compounds also provide a path for the production of novel plant derivatives with enhanced bioavailability, as has been described for isoflavonoids. 12The use of nanocarriers also increase bioavailability and can deliver the metabolite to its specific target point.Microbial SynBio can contribute to human health and well-being by enhancing bioavailability, following the pathways described below.

Enhancing pharmacokinetics properties through molecular modifications
Molecular modifications such as glycolizations and methylations can be used to enhance a compound's bioavailability without altering its intended effects.These modifications can be performed using microbial SynBio with the downstream addition of one or two steps within the established metabolic pathway.For instance, oxygen and carbon glycolizations and methylations improve the pharmacokinetic properties of flavonoids.These flavonoid F Rojo et al.
Perspective: Microbial synthetic biology for plant metabolite production compounds, which usually have low solubility and are easily metabolized or excreted by the human body, can be upgraded with the addition of sugars or methyl groups to help mitigate the issues surrounding their suboptimal bioavailability. 44

Nanocarriers
Another alternative to increase bioavailability is the use of nanocarriers as a delivery system.Once the plant compound is produced and purified it can be added to different carriers to expand its use for clinical applications.Different types of nanocarriers have been described for plant products: organic (lipid-based), biopolymer, and inorganic (metal/ magnetic nanoparticles). 45Nanoparticles can be conjugated with different ligands, such as antibodies, peptides, aptamers, or even small molecules to deliver the effector molecule to a specific targeted site. 46These carriers can be used to both avoid degradation (increasing bioavailability), and to stimulate the necessary chemical action within the target physiological zone, enhancing the therapeutic efficacy of the delivered compound.Isolated cells are also explored as delivery systems, tagging the cell membrane with antibodies to generate the desire action within a tissue-specific target site. 47

Discussion
To utilize the full potential that microbial SynBio has to offer, the knowledge we have developed as a scientific community must be applied towards sustainable production methods that reconcile both human and environmental well-being.Advances in next-generation sequencing and bioinformatics allow us to understand better how plant molecules are biosynthesized.Molecular engineering tools are constantly being improved, expanding the limits of what we can produce.However, these advances need to come with the understanding that progress should be tempered with sustainability, and that the goals of microbial SynBio should look beyond the economic value of the end product.We propose to perform ecological and economic analysis towards a generation of circular bio-economies.In this regard, existing industries are facing pressure to become a sustainable business, where the framework of the policies and regulations are still in a developing stage worldwide.Instead of just focusing efforts to transform existing industries into more sustainable ventures, the concept of a bioeconomic approach should also be encouraged at the outset of business creation.In that sense, the whole production cycle should be designed to consider circular bio-based solutions that address climate neutrality.Moreover, the process should be enhanced not only with a focus on economic indicators such as production cost and yield, but also social acceptance.Would consumers consider how a plant-based drug is produced when deciding what to buy?In our opinion, these considerations will also be applied to the nutraceutical and pharmaceutical industries in coming years given the changing purchasing behavior of consumers to favor environmentally friendly alternatives.
The application of microbial SynBio to produce plant bioproducts still face challenges.Negative feedback from intermediate molecules along the metabolic pathway is one of the major reasons for the achievement of lower yields than expected.The stress that the chassis goes through due to the incorporation of multiple genes also creates a metabolic burden that needs to be compensated, for instance using co-culture strategies.Those strategies are not always easy to optimize because factors such as pathway breakdown and strain growth balance are challenging to moderate.The presence of growth inhibitors also needs to be tested for, and the chassis should be designed to tolerate the action of inhibitory compounds.It is also important to consider the potential challenges associated with post-transcriptional and post-translational modifications of genes and enzymes involved in biosynthesis.Many plant-derived proteins undergo intricate modifications that are specific to their native organisms, including glycosylation, acylation, and phosphorylation, among others. 48These modifications can play a crucial role in the bioactivity, stability, and bioavailability of the target compounds.When transferring biosynthetic pathways to microbial hosts, these native modifications may be absent or altered, leading to potential differences in the final product.It is therefore essential to engineer the microbial chassis to incorporate or mimic the machinery from native hosts.Recent advancements in synthetic biology have facilitated the development of strategies to introduce or reconstitute these modifications in microbial hosts.For instance, the utilization of CRISPR-Cas9based genome editing techniques has allowed the precise integration of genes encoding enzymes involved in specific post-translational modifications. 49rom a bio-economic perspective, more effort should be made towards waste-consuming microorganisms as a production platform to develop a truly circular economy.Novel microbes and renewable substrates alternatives need to be further explored and characterized to reduce feedstock costs in microbial-based sustainable industries.Up-to-date, engineering tools and scale-up strategies for non-model systems are still scarce.Collective efforts should be implemented for the identification, whole sequencing, and establishment of novel chassis that can act as third-generation biorefineries.The comparison between the in planta and ex planta production systems requires further analysis on a caseby-case basis.Microbial-based systems do not always surpass native host production, as is the case for 4-hydroxybenzoic acid, with a higher accumulation rate and consequently lower production cost when using a native host. 50From our perspective, the benefits of ex planta production systems will only outweigh current plant-based systems if microbial-based innovations are engineered to reduce manufacturing costs consistently, improve bioproduct yields, and address ongoing ecological and socio-economic issues effectively.

Conclusion
It is essential to expand the application of sustainable production systems towards a circular economy, where waste residues from human activities can be exploited as substrate sources.We highlight the reasons why sustainable production systems should be promoted by using smart microbes as bio-factories for producing plant metabolites with positive impacts on human health.Microbial SynBio solutions have shown to be more efficient than plant-based production, and can use fewer resources (land, water, fertilizers, etc.).Moreover, the implementation of new techniques such as combinatorial biosynthesis and co-culture systems, have been shown to increase the yield of plant metabolites.Even in cases where the full metabolic pathway is not described, in-silico analysis such as machine learning, protein modeling, and retro-pathing can be applied to resolve the biosynthetic potential of a microbial candidate.Furthermore, more environmentally friendly chassis must be explored to make the production of plant derivatives sustainable, such as using C1/C2 molecules as carbon sources, and/ or residues considered waste from human activities for achieving greener solutions.Finally, microbial SynBio can be used to apply molecular modifications to candidate compounds by adding steps into established biosynthetic pathways, creating improved plant derivatives for clinical applications towards the realization of the UN Sustainable Development Goals.

Figure 1 .F
Figure 1.Schematic representation of sustainable microbial-based production systems.

F
Rojo et al.Perspective: Microbial synthetic biology for plant metabolite production 1493

Table 1 .
Another example of microbial metabolic plasticity is a study that utilized the methanol oxidation pathway from P. pastoris incorporated into S. cerevisiae, enabling a high methanol consumption rate for pyruvate production. 33 Rojo et al.Perspective: Microbial synthetic biology for plant metabolite production1489 Example of production systems using different strategies, chassis and substrate sources.Section 1: Model organisms using single or polycultures and combinatorial biosynthesis.Section 2: Microbes using sustainable feedstocks.Section 3: Third generation biorefineries using CO 2 as a carbon source.