Wei-Chih
Chin
ab,
Yang-Zhi
Zhou
ab,
Hao-Yung
Wang
abc,
Yu-Ting
Feng
ab,
Ru-Yin
Yang
ab,
Zih-Fang
Huang
ab and
Yu-Liang
Yang
*ab
aAgricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan. E-mail: ylyang@gate.sinica.edu.tw
bBiotechnology Center in Southern Taiwan, Academia Sinica, Tainan, Taiwan
cDepartment of Wood Based Materials and Design, National Chiayi University, Chiayi, Taiwan
First published on 29th January 2024
Covering: up to 2023
Conjugated polyynes are natural compounds characterized by alternating single and triple carbon–carbon bonds, endowing them with distinct physicochemical traits and a range of biological activities. While traditionally sourced mainly from plants, recent investigations have revealed many compounds originating from bacterial strains. This review synthesizes current research on bacterial-derived conjugated polyynes, delving into their biosynthetic routes, underscoring the variety in their molecular structures, and examining their potential applications in biotechnology. Additionally, we outline future directions for metabolic and protein engineering to establish more robust and stable platforms for their production.
Traditional sources of polyacetylenes include plants from families like Apiaceae and Asteraceae,1,3 marine organisms such as algae and sponges,5 and fungi like Hydnum repandum L.12 Chemical synthesis has also been attempted, but the inherent instability of acetylenes and their intermediates often hinders successful synthesis.13 In recent decades, there has been a growing interest in exploring the microbial world for its potential biotechnological applications, including the discovery of novel compounds like polyynes.14 Polyynes, in particular, have been the subject of multiple studies aimed at characterizing their molecular diversity, ecological roles, evolution and origins. The first bacterial polyynes, cepacins A (6) and B (7),15 were discovered in the 1980s from Pseudomonas cepacia (later reclassified to Burkholderia diffusa).14,16 Since then, several other bacterial polyynes like caryoynencin (8),17 collimonins A–D (9–12),18 ergoynes A and B (13, 14),19 and protegenins A–D (15–18, syn. protegencins) have been identified,14 but the biosynthetic gene clusters (BGCs) responsible for their production remained elusive until more recent studies employed techniques like transposon mutagenesis and genome mining.16,20
Bacterial polyynes are becoming an increasingly important area of research due to their varied bioactivities and biosynthetic pathways. Compared to plants and eukaryotes, the biosynthesis genes in bacteria tend to be clustered, simplifying the study of their production and the comparative analysis of modifications across different bacterial strains. The development of bacterial genomic assembly techniques has transformed our understanding of bacterial evolution and greatly advanced the discovery of natural products, particularly through phylogeny-led genome mining methods.21 These techniques have enabled the identification of novel metabolic products, including those from BGCs responsible for polyyne biosynthesis.14,22,23
This review aims to cover a broad spectrum of topics related to bacterial polyynes, including their history of discovery, isolation, biological activities, ecological roles, and the BGCs involved in their production. Additionally, it will discuss the enzymatic pathways involved in polyyne biosynthesis and offer insights into future research directions in this field.
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Fig. 1 Structural diversity of acetylenes and bacterial-derived polyynes. Falcarindiol (1) and falcarinol (2) were discovered in carrots,6 while peyssonenynes A and B (3, 4) were identified in Peyssonnelia caulifera,15 dihydroxerulin (5) was identified in Xerula melanotricha (yellow region).92 Fischerellins A and B (19, 20) were found exclusively in F. muscicola,29,93 while ergoynes A and B (13, 14) were identified in G. sunshinyii (gray region).19 Bacterial polyynes are classified based on their carbon backbone into two categories: those with a 16-carbon backbone (blue region) and those with an 18-carbon backbone (red region). Collimonins C and D (11, 12) and massilins A–C (22–24) were discovered in Massilia sp. YMA4,23 collimonins A–D (9–12) in C. fungivorans Ter331,18 cepacins A and B (6, 7) in B. diffusa and B. ambifaria BCC0191,15,37 Sch 31828 (23) in Microbispora sp. SCC1438,25 and protegenin A (15, syn. protegencin) in P. protegens strains Cab57,22 Pf-5 and CHA0.14 Protegenins B–D (16–18) were identified explicitly in P. protegens Cab57,22 while caryoynencin (8) was isolated from T. caryophylli Ballard 720 and B. gladioli BSR3.16 |
Ethyl acetate (EtOAc) is frequently employed for initial extraction due to its high affinity for nonpolar and moderately polar compounds. Given that polyynes are often long-chain carbons and polyalkynes, they are typically nonpolar or moderately polar and lipophilic, making them well-suited for extraction with EtOAc.18,20,23 Polyynes with a 16-carbon backbone are more hydroxylated than those with an 18-carbon backbone, complicating the determination of their structure and stereo-configuration. Two primary methods are used for isolating polyynes for further analysis. The first involves using dimethyl sulfoxide (DMSO) or dimethylformamide especially for hydrophobic polyynes to inhibit degradation.14,18,23,26 The solution is then concentrated and subjected to reverse-phase column liquid chromatography for qualitative analysis, although this method offers only semi-quantitative results. The second approach uses click chemistry to stabilize the terminal alkyne, facilitating qualitative and quantitative analyses.16,26,27 However, this technique alters the natural structure and biological function of the polyyne.
Addressing polyyne instability is essential, particularly for large-scale preparations. Traditional storage solutions like DMSO are not cost-effective for extensive metabolic engineering projects. Alternative, more economical options such as palm oil are recommended for extraction and structural protection, with the advantage of serving as a xenogeneic carbon source for bacterial cultivation. Future research should also investigate alternative click chemistry methods that maintain the biological activity of the polyyne. One promising direction is using halogens as chemo-selective biocatalysts to form haloalkynes, which may preserve biological activity after modification.28 Therefore, optimizing extraction and isolation protocols and identifying stable, cost-effective solutions are critical areas for future research in the production and application of bacterial polyynes.
Sch 31828 (21) from Microbispora sp., also known as triyne carbonate (L-660,631), is unique in its antifungal activity against Candida albicans and antibacterial activity against Gram-positive bacteria.31,32 Collimonins are primarily antifungal agents effective against Aspergillus niger, with collimonins A, C, and D (9, 11, 12) also exhibiting hyphal branching activities.18,20
Protegenins (15–18, syn. protegencins) exhibit antioomycete and antifungal activities, targeting organisms like Pythium ultimum and Fusarium oxysporum.22 Massilins A–C (22–24) and collimonins C and D (11, 12) target ERG10 to exert antifungal activity against C. albicans.23
The mechanisms of action for bacterial polyynes are still not fully understood, but some insights have been gained.23,31–33 For example, Sch 31828 (21) has been shown to inhibit sterol biosynthesis in both C. albicans and human liver Hep G2 cells by interfering with the mevalonate pathway, essential for cholesterol synthesis. This compound affects the viability of C. albicans cells and inhibits the incorporation of acetate and octanoate into sterol in Hep G2 cells.31–33 Similarly, polyynes derived from Massilia sp. YMA4 have been shown to possess antifungal properties against both C. albicans and fluconazole-resistant Candida species.23 The resistance gene, masL, has been located within the BGC of these polyynes.23 This particular gene encodes for the enzyme acetyl-CoA acetyltransferase (ACAT), and its expression in polyyne-sensitive strains of C. albicans effectively counteracts the inhibitory action of polyynes.23 Further insights from crystallographic analysis have established that these bacterial polyynes are covalent inhibitors of ACAT.23 They compromise cell membrane integrity and hinder cellular viability by targeting the ACAT of C. albicans, ERG10, a pivotal enzyme in the ergosterol biosynthesis pathway essential for the formation of fungal cell membranes and a promising target for antifungal drugs. A related study on Aspergillus fumigatus revealed that diminished expression of the analogous gene, erg10A, elevates susceptibility to oxidative stress and agents that perturb the cell wall. This implies bacterial polyynes impede fungal growth by disrupting ergosterol biosynthesis.34,35
Bacteria polyynes serve crucial ecological functions, particularly in providing defense mechanisms for various organisms. In the context of plants and insects, polyynes act as potent antimicrobial agents that protect against harmful microbes. For instance, cepacin A (6) and caryoynencin (8) have been shown to protect germinating crops against Pythium damping-off disease.36,37 Protegenins (15–18, syn. protegencins), the active metabolites of P. protegens strains Pf-5 and CHA0, have been identified as a critical factor contributing to their outstanding performance as biocontrol agents against plant pathogens.14,22,37 Fischerellin A (19) inhibits the growth of various plant diseases, such as brown rust and powdery mildew, at different concentrations.29 In light of the growing necessity to minimize reliance on synthetic pesticides, the adoption of biological control agents sourced from species like Burkholderia ambifaria,36Burkholderia gladioli,38 and Pseudomonas protegens,22 offers a viable, sustainable method for crop protection.22,36,37 However, given the potential pathogenic risks associated with Burkholderia species,36,38 a promising solution lies in transferring BGCs from these organisms into non-pathogenic hosts.39 This strategy enables the development of safe biocontrol agents while mitigating the risks of opportunistic infections. Bacterial polyynes also play a defensive role in bacterial symbionts. For example, caryoynencin (8), produced by symbiotic B. gladioli, protects the eggs of certain herbivorous beetles from bacterial infections. This bacterium also produces other novel antimicrobial compounds like lagriene and sinapigladioside, further emphasizing their role as protective agents.38,40
In addition to their defensive roles, bacterial polyynes are involved in microbial interaction by manipulating nutrient availability or disrupting signaling systems. For example, protegenin A (15, syn. protegencin) released by P. protegens infiltrates algal cells and degrades carotenoids in their eyespot, effectively inhibiting the phototactic behavior of Chlamydomonas reinhardtii.41 This is further facilitated by the secretion of another compound, orfamide A, which induces deflagellation and morphological changes in the alga.42 These findings underscore the multifaceted ecological roles of bacterial polyynes, from serving as natural biocontrol agents in agriculture to acting as chemical mediators in microbial communities.
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Fig. 2 Phylogram of confirmed polyyne BGCs in bacteria. Homologous genes within each cluster were identified using cblaster.50 Subsequently, multiple sequence alignments (MSAs) were generated using MUSCLE,54 the distance (maximum likelihood, ML, bootstrap 5000 times) between 64 bacteria and phylogenetically informative regions were trimmed using TrimAl.53 The resulting phylogeny was constructed using Bayesian computation with Markov Chain Monte Carlo (MCMC) sampling, employing MrBayes,52 and the visualization was accomplished using iTOL.51 Species within the blue box are known to produce C16 polyynes, while those within the red box are documented as producers of C18 polyynes. The yellow strip represents Betaproteobacteria, and the green strip represents Gammaproteobacteria. |
Studies involving transcriptomics analysis, single-gene mutation or expression experiments, heterologous reconstitution, and BGC comparisons have been instrumental in identifying the essential genes required for polyyne biosynthesis.14,22,23,43 These BGCs commonly feature a set of conserved core genes. These genes encode for a fatty acyl-AMP ligase (FAAL), three fatty acid desaturases (FAD), an acyl carrier protein (ACP), a hydrolase/thioesterase (H/TE), and an electron transport component such as ferritin or rubredoxin (Rd). Distinct from plant-derived alkynes, bacterial polyynes operate on a thiotemplate mechanism, necessitating the loading of fatty acids onto a specialized ACP. The role of desaturases is especially significant, as they have the capability to introduce multiple triple bonds into the fatty acid chain. The inclusion of an additional electron transport component has been shown to boost polyyne production substantially.23,43
In addition to these core genes, polyyne BGCs may encode enzymes for further structure modifications. These tailoring enzymes include monooxygenases like cayG or dioxygenases such as masB or ccnG, which are responsible for the hydroxylation of polyynes.23 Cytochrome P450 monooxygenases (CYP) can introduce an allylic hydroxy moiety, with cayK and cayL identified as rare designated CYP redox partners (Fig. 3).44 These findings suggest that the bacterial genome may already contain the instructions for various potential polyyne modifications, meanwhile providing us opportunities for the engineering of polyyne structures to enhance their chemical diversity.
Antibiotic-producing bacteria often come equipped with self-protection mechanisms, frequently encoded within their BGCs. The discovery of the resistance gene masL in the massilins BGC (mas) is an example that also leads to identifying the antifungal target.23 Efflux transport systems also contribute to bacterial self-protection. Although the precise export mechanisms for bacterial polyynes are not yet confirmed, their BGCs encode efflux transporters such as a major facilitator superfamily (MFS) transporter in the cepacins BGC (ccn)36 and a resistance-nodulation-cell-division superfamily (RND) efflux system in the cay BGC,16 suggesting their involvement in secretion (Fig. 3).
Evolutionarily, the ccn BGC branched into two distinct BGCs: mas BGC and collimonins BGC (col). Each evolved to contain its own unique self-protection mechanism (Fig. 2 and 3). The mas BGC and its ancestral ccn BGC include an ACAT gene, which is absent in the col BGC. On the other hand, both the ccn BGC and col BGC feature an efflux transport gene, missing in the mas BGC. Interestingly, in C. albicans, the upregulation of ACAT genes (masL and ERG10) counteracts the antifungal effects of collimonins C (11), D (12), and massilin A (22). However, this same upregulation of ACAT genes does not confer resistance against massilin C (24).23 This is significant, given that all these compounds act as ACAT inhibitors. The absence of massilin C (24) in its native host—due to its efficient conversion into collimonins C (11), D (12), and massilin A (22)—suggests that a singular self-defense mechanism may suffice for Massilia sp. YMA4. Conversely, massilin C (24) suppresses the growth of C. albicans through an ACAT-independent route.
Bioinformatics and gene cluster comparisons have identified three conserved genes for membrane-bound fatty acid desaturases (Fig. 3b).14,23,43 These desaturase genes are connected to jamB, which plays a role in forming the terminal alkyne component of jamaicamide B in marine cyanobacteria.45 Point mutations in the active residues of each desaturase led to the cessation of polyyne production, confirming that a complete set of fully functional desaturases is essential for polyyne synthesis. This also demonstrates the conserved histidine regions in the desaturase genes coordinating di-iron centers at the active site.43
Bacterial polyyne biosynthesis is characterized by four main stages: initiation (ACP ligation), desaturation, ACP release, and possible additional modifications. Each phase is driven by specific enzymes and processes, as revealed by the functional dissection and reconstruction of polyyne gene clusters in heterologous hosts, illustrated in Fig. 4. Recent research has suggested two potential biosynthetic precursors for protegenin (syn. protegencin) and caryoynencin as unsaturated fatty acids (UFA)43,44 or saturated fatty acids (SFA).26 These insights are crucial for future bioengineering projects. However, challenges such as managing multi-gene transformations, organizing gene cassettes, regulating individual genes, and ensuring effective cross-species integration remain. These factors are essential for the successful manipulation of bacterial polyyne production.
Polyyne production in bacteria involves a complex interaction of BGCs, the genetic background of the host species, and diverse culture conditions. The success of heterologous BGC expression depends on several factors, including the design of the genetic constructs, the composition of the growth media, and the chosen host species.46 For example, cepacin and caryoynencin BGCs from various Burkholderia strains were effectively expressed in Paraburkholderia, a non-pathogenic host, using a yeast-adapted Burkholderia–Escherichia shuttle vector.39,47 Culture conditions, particularly temperature and pH, also play a significant role in polyyne biosynthesis. The optimal growth temperature for the heterologous host is crucial; for instance, Paraburkholderia strains showed a preference for growing at 30 °C compared to 37 °C.39 A higher cell density can indirectly boost polyyne production. Furthermore, changes in pH have been observed to increase the production of cepacin A (6) in B. ambifaria BCC0191 but decrease caryoynencin (8) production in B. gladioli BCC1697.39
Media composition further adds a layer of complexity to the regulation of polyyne biosynthesis. For example, more significant quantities of cepacin A (6) and caryoynencin (8) were produced when B. ambifaria BCC0191 and B. gladioli BCC1697 were grown on basal minimal media as compared to pea exudate media.39 An antagonism assay of Massilia sp. YMA4 showed that antifungal metabolites were produced in potato dextrose agar but not in yeast malt agar, revealing the role of media in regulating polyyne production.23 Moreover, the presence of different carbon and nitrogen sources in the culture media can lead to variations in microbial metabolic products. For example, glucose in the culture conditions enhances the fatty acid synthesis system, while long-chain fatty acids promote the fatty acid degradation system, which is essential for cell growth.48,49 These findings collectively suggest that the regulation and expression of polyyne BGCs can vary significantly across different bacterial strains and culture conditions.
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Fig. 5 Cladogram of potential polyyne BGCs in bacteria. Homologous genes within each cluster were identified through cblaster using the BGC from Massilia sp. YMA4 as query sequence.50 Subsequently, multiple sequence alignments (MSAs) were generated using MUSCLE,54 and phylogenetically informative regions were trimmed using TrimAl.53 The resulting phylogeny was constructed using Bayesian computation with Markov Chain Monte Carlo (MCMC) sampling, employing MrBayes,52 and the visualization was accomplished using iTOL.51 Colored regions on the branches indicate the type of the polyyne backbone. Colored strips outside the graph are the defended groups for the clusters. The highlighted bold branches are listed as follows: black, clusters with predicted masB homolog; violet, Streptomyces rochei 7434AN4 (AP018517.1); magenta, Streptomyces sp. V1I6 (NZ_JAUSYR010000001.1); red, Eleftheria terrae P9846-PB (NZ_CP106951.1); blue, Pyxidicoccus xibeiensis QH1ED-7-1 (NZ_JAJVKV010000003.1); green, Gynuella sunshinyii YC6258 (NZ_CP007142.1); brown, Sphingomonas spp. (NZ_JAVIZP010000001.1 and NZ_JAVIYV010000001.1). |
The initial division is the monophyletic group with a 16-carbon backbone, segmented into two subsets: the Oxalobacteraceae group and Burkholderia group I. The first subset contains three genera—Collimonas, Massilia, and Pseudoduganella—while the second encompasses four principal Burkholderia lineages, namely B. ambifaria, B. contaminans, B. stagnalis, and B. vietnamiensis. Additionally, we observe that masB homologs are limited to 297 clusters within this division (bold black branches), suggesting that masB homologs likely engage solely in the hydroxylation of 16-carbon backbone polyynes.
The second division comprises lineages stemming from actinomycetes. A majority of the clusters within this section are attributed to Streptomyces, accounting for their notable similarity. Nevertheless, two unique instances stand out. The first is an isolated strain from wheat rhizosphere, designated as Streptomyces sp. V1I6 (Fig. 5, bold magenta branch);55 this represents the sole instance we discover featuring a FAAL–FAD fusion protein (Fig. 6b). The second unique instance is linked to a historic strain, Streptomyces rochei 7434AN4 (Fig. 5, bold violet branch), known for producing lankacidin and lankamycin since the previous century.56 Although this strain has been extensively researched and is anticipated to generate a wide array of bioactive compounds,57 we identify two unreported, complete putative polyyne BGCs.
The final division is a paraphyletic group characterized by 18-carbon backbone polyyne, and it consists of three subcategories: the Trinickia group, Burkholderia group II, and the Pseudomonas group. When contrasted with the 16-carbon backbone group, these subgroups exhibit reduced diversity in taxonomic structure and cluster architectures. In essence, the clusters identified here closely align with those previously reported (Fig. 3).
Intriguingly, our analysis also reveals five orphan lineages at the base of this partition, which we hypothesize to be the result of horizontal gene transfer, as these lineages are part of four different classes within four distinct genera of Pseudomonadota. Of these, only Gynuella sunshinyii YC6258 has been previously examined and identified as a producer of ergoynes (13, 14) (Fig. 5, bold green branch). This particular strain was isolated from the root tissue of the halophyte Carex scabrifolia and is suggested to possess potent antifungal activity.58 Interestingly, ergoynes differ structurally from other known bacterial polyynes. It is believed to be formed via a non-enzymatic reaction between a tetrayne precursor (25) and ergothioneine (26), as evidenced by the separate BGCs for ergothioneine and polyyne in the G. sunshinyii genome (Fig. 6a).19 To explore broader connections in polyyne biosynthesis within Gammaproteobacteria, we conducted a comparative analysis of gene content between the published pgn BGC from P. protegens Pf-5 and G. sunshinyii (Fig. 6b). The G. sunshinyii BGC contains conserved core genes coding for fatty acyl-AMP ligase, two types of fusion desaturases, an acyl carrier protein (ACP), a fusion protein of desaturase and hydrolase, and a rubredoxin.14 These findings indicate that the tetrayne precursor (25) varies from other C18 fatty acid sources like stearic or vaccenic acids, suggesting the presence of other diverse polyynes in nature worth investigating. Additionally, beyond the Pseudomonas group and G. sunshinyii, we discover two Sphingomonas isolates originating from sorghum phyllosphere (Fig. 5, bold brown branch) that possess clusters of a FAD–H fusion protein (Fig. 6b). In summary, Pseudomonas, Gynuella, and Sphingomonas represent the only known lineages to harbor FAD–H fusion polyyne BGCs. However, the origins and reasons for this unique genetic configuration remain enigmatic.
The remaining two clusters are associated with bacterial strains known for their antimicrobial properties. Eleftheria terrae P9846-PB, known for producing teixobactin, was sourced from a grassy field using the iChip isolation technique (Fig. 5, bold red branch).59 Although its putative polyyne BGC contains all six core genes, neither its functional role nor its ability to synthesize polyyne has been verified. On the other hand, Pyxidicoccus xibeiensis QH1ED-7-1, a member of the myxobacteria group renowned for complex social behaviors and generating a wide array of bioactive metabolites,60,61 was isolated from forest soil. While multiple BGCs were predicted using antiSMASH,62 none have been attributed to polyyne synthesis. Interestingly, the putative polyyne BGC in strain QH1ED-7-1 features duplicated FAAL and ACP genes (Fig. 6b). Given the predatory nature of myxobacteria and the roles of FAAL and ACP, we hypothesize that this gene duplication may be a strategic adaptation by the strain to amplify its precursor pool for polyyne-like compound biosynthesis, thereby enhancing predation efficiency. Our analytical pipeline is capable of identifying not just the well-characterized but also the cryptic polyyne BGCs. Nevertheless, it's worth noting that using Massilia sp. YMA4's BGC as a query sequence may introduce a selection bias during database searches. For the putative BGCs, especially those in the actinomycetes group, additional empirical studies are needed to substantiate our hypotheses.
In addition to their antifungal properties, bacterial polyynes offer a new approach to treating hyperlipidemia, a significant global health issue. While statins are commonly used to inhibit HMG-CoA reductase in lipid metabolism, they often fail to reduce low-density lipoprotein cholesterol levels in many patients adequately. By targeting ACAT, bacterial polyynes provide an alternative avenue for hypolipidemic treatment.63–65
Moreover, bacterial polyynes could have far-reaching implications in the treatment of human diseases like cancer and Alzheimer's. The mevalonate pathway and its metabolites are also essential for the growth and malignant progression of various cancers, including prostate, breast, lung, and liver. ACAT1, the mitochondrial acetyl-CoA acetyltransferase, has been suggested as a potential therapeutic target for cancer.66,67 ACAT inhibitors have also been shown to inhibit the generation of beta-amyloid peptides, a hallmark of Alzheimer's disease, suggesting their potential utility in treating this condition.68,69 However, like polyacetylenes from plant and eukaryote sources, the cytotoxicity of bacterial polyynes is a significant factor to consider. Studies have shown that some polyacetylenes exhibit cytotoxic effects on cancer cell lines while having minimal or no impact on normal cells, and this has been further explored in animal experiments.70–73 Despite these promising attributes, the discussion on the side effects of polyynes is limited. Safety in therapeutic applications remains an area requiring more extensive research for a thorough understanding. Consequently, while there are high hopes for the therapeutic potential of polyynes, their use necessitates further investigation to fully comprehend their benefits and risks.
Beyond human health, bacterial polyynes have also been explored for pest insect control, where they impair tick reproductive ability and egg development. This suggests their untapped potential in various fields. Therefore, the structural diversity of bacterial polyynes and their associated biosynthetic gene clusters could open new avenues for drug development across a range of medical conditions.74
The potential of microorganisms in drug discovery has been acknowledged for nearly a century, yielding a wide range of antibiotics, anticancer agents, and cholesterol-lowering drugs. However, the natural production of polyynes often faces challenges such as low yield and culture condition specificity. Advances in genome sequencing and synthetic biology have reinvigorated interest in microbial metabolites for pharmaceutical applications. Genome mining techniques have identified potential polyyne producers based on the similarity of their BGCs to known strains. Furthermore, synthetic biology tools like protein engineering and DNA synthesis offer avenues for reprogramming microbial metabolic systems to enhance the productivity of target metabolites.
Technological advancements in synthetic biology and genetic engineering, including CRISPR/Cas systems and modular DNA assembly methods, have revolutionized microbial cell engineering.75,76 These tools enable the construction of metabolic pathways and biological circuits that control cellular behavior, which is particularly useful for organisms that are not easily cultured or for compounds identified through metagenomic datasets.77 Preferred host microorganisms like E. coli and Saccharomyces cerevisiae benefit from mature techniques such as gene editing and large-scale fermentation.46,78,79 Additionally, recent developments in dynamic sensor-regulator systems optimize substrate concentration and timing, critical factors in industrial fermentation.
Metabolic engineering techniques have been specifically applied to optimize the production of bacterial polyynes (Fig. 7). A focus on precursor carbon flow and the careful design of host fermentation conditions can significantly enhance bacterial polyyne yields. Recent articles have described the possible precursors for protegenin (syn. protegencin)/caryoynencin biosynthesis as UFA (elaidic acid)43 and SFA (stearic acid),26 emphasizing the need for precise control over fatty acid metabolism.26,43 Techniques like molecular cloning, gene deletion, and overexpression have been employed to manipulate fatty acid metabolism.80 For instance, the manipulation of specific genes, such as fabA and des, can modify the abundance of both saturated and unsaturated fatty acids, consequently influencing cellular properties like ethanol controlling fabB expression.81–83 Moreover, when fabH, fabF, or both genes are overexpressed in E. coli BL21 (DE3), it leads to a reduction in fatty acid synthesis and an elevation in cellular malonyl-CoA levels.84 Notably, the deletion of fabF has the specific effect of inhibiting the production of C18:1 and C18:0 fatty acids.85 In the context of fatty acid degradation, fadD, fadR deletion, and overexpressing fadL, fadE, fadB, and fadA in E. coli serve to increase the productivity of hydroxy long-chain fatty acids.78,86 However, it's worth noting that heterologous overexpression of the polyyne BGC may limit polyyne production, potentially due to competition for fatty acids or the accumulation of polyyne. An interesting strategy for enhancing the host organism's tolerance to polyyne involves leveraging the presence of efflux pumps or self-resistance genes within some BGCs.39,87–91
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Fig. 7 Efficient strategy for increasing the biosynthesis of microbial polyyne. Polyyne BGCs = polyyne biosynthetic gene clusters. |
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