Engineering cyanobacteria for the production of aromatic natural products

Aromatic natural products are important for improving human health and quality of life. Large-scale availability of these compounds from plants is limited by low yield and cumbersome extraction. Building high-performance microbial cell factories to produce aromatic natural products by means of metabolic engineering and synthetic biology is a viable option. In the context of climate change and global resource scarcity, choosing solar-powered and carbon-fixing microbial cyanobacteria instead of chemical heterotrophic microorganisms to produce aromatic compounds might be a more progressive and better solution. In this review, we summarize the principal strategies for improving the production of aromatic natural products in engineered cyanobacteria, including regulation of metabolism, optimization of synthetic pathway, culture process development, and microbial cooperation, high-lighting the potential and availability of this microbe as a novel chassis cell.

Aromatic natural products have unique biological activities and are of great significance to human production and life [2,3,[5][6][7].Phenolic acids have a variety of bioactivities and are also the precursors of some valueadded compounds and drugs [1].p-Coumaric acid is an important platform chemical for synthesizing other phenolic compounds [8].Caffeic acid has antiviral activity against influenza virus, and its derivative caffeic acid ester can suppress colon cancer cells [2,9].Flavonoids have been shown to eliminate macromolecular damage and reduce cell senescence.For example, naringin can increase the activity of antioxidant enzymes [10], and apigenin can remove excess free radicals [11,12].Resveratrol, a stilbenoid compound, can improve cardiovascular function by modifying vascular function and attenuating lipid accumulation, demonstrating great potential in the pharmaceutical field [13][14][15].Coumarins are a class of natural agents with a wide range of action targets [16].Due to the antithrombotic activity and low toxicity of coumarins, coumarin-based drugs (e.g., warfarin) have been used clinically to treat thrombosis [17].In addition to medicine and pharmaceutical fields, aromatic compounds also play an important role in the food field, which can shape human food preferences and eating behaviors by influencing food flavor [18].The aromatic compounds responsible for the aroma of plants, such as vanillin (vanilla), phenylacetaldehyde (rose) and cinnamaldehyde (cinnamon), are in high demand in the food industries.According to a new research report announced by Global Market Insights Inc., the aroma chemical market size is estimated to reach USD 9 billion by 2032.Notably, the rising focus on eco-friendly products will spur natural source segment growth, and the natural segment is slated to record a valuation of more than USD 4 billion by 2032 [19].
A wide variety of aromatic natural products exist widely in plants, assisting plants to complete physiological processes such as growth and development, resistance to ultraviolet radiation and defense against pathogen invasion [20].The synthetic pathways and flux regulation in plants have been extensively studied [21].However, plants are generally not good sources for large-scale production of aromatic chemicals as a result of the limitations of the growing season and laborious extraction processes [18].Microorganisms have become attractive platforms for the synthesis of aromatic compounds because of their ability to grow rapidly and accumulate high biomass under suitable culture conditions, as well as their easy genetic modification procedures.Benefiting from advances in metabolic engineering and synthetic biology, the project of heterologous production of aromatic compounds by microorganisms has developed rapidly.Several aromatic compounds synthesized by microbes, such as vanillin and resveratrol, have reached commercial-scale production [1,22].However, for heterotrophic chassis, such as Escherichia coli (E.coli) and Saccharomyces cerevisiae (S. cerevisiae), the high-cost sugar-based feedstock requirements reduce the economic viability and sustainability of production [23,24].In the time of climate change and global resource shortage, producing basic and fine chemicals in a CO 2 -neutral manner is the key to achieving true sustainability [25].Cyanobacteria combine the characteristics and benefits of plants and microbes, making them strong candidates for new cell factories.As photosynthetic autotrophs, cyanobacteria can convert CO 2 and water into various chemical compounds driven by solar energy, without the need for carbon feedstocks.Compared to plants, cyanobacteria have a faster growth rate and a more efficient ability to capture solar energy [26].In terms of physiological structure, cyanobacteria have abundant membrane structures and cytochromes, which improves the compatibility of expressing a variety of membrane-binding synthases of plant origin.Plentiful NADPH and ATP in cyanobacteria provide sufficient reducing power and energy for the synthesis of plant metabolites [27].These properties, together with relatively simple genetic manipulation, make cyanobacteria ideal chassis for the sustainable production of aromatic natural products (Fig. 1) [28][29][30].

Regulation of metabolism Elimination of regulatory mechanisms
There is complex and strict regulation of shikimate and AAAs synthesis pathways in cells, including the regulation of positive and negative feedback and transcriptional regulatory factors [56,57].DAHPS is a key enzyme that controls carbon flux into the shikimate pathway, receiving strong allosteric feedback inhibition from specific aromatic amino acids in vivo [58][59][60].In E. coli, three DAHPS isoenzymes encoded by aroG, aroF, and aroH, are shown to be feedback-inhibited by ʟ-Phe, ʟ-Tyr, and ʟ-Trp, respectively.Eighty percent of the DAHPS activity in E. coli is attributed to AroG [61,62].In S. cerevisiae, two DAHPS isoenzymes encoded by the ARO3 and ARO4 genes are feedback inhibited by ʟ-Phe and ʟ-Tyr, respectively [63,64].The activity of DAHPS has been shown to be inhibited by ʟ-Phe or ʟ-Tyr in more than half of the 48 tested cyanobacteria strains [65,66].Moreover, the metabolism of CHA, a precursor of AAAs synthesis, is also strictly controlled.The activities of CM-PDH, CM-PD complexes and AS in E. coli are inhibited by ʟ-Tyr, ʟ-Phe and ʟ-Trp, respectively [67][68][69].The activity of CM can be inhibited by ʟ-Tyr or ʟ-Phe in a few cyanobacterial strains, such as Synechococcus sp.27180 [66].The activity of PD is inhibited by ʟ-Phe in Synechocystis sp.29108 [65].Substitution of specific amino acids can reduce or eliminate allosteric feedback inhibition of the substrate, resulting in a feedback-inhibition resistant (fbr) enzyme.These single amino acid mutants of AroG, such as Asp146Aen, Pro150Leu, Leu175Asp, Leu179Ala, Ser180Phe, Phe209Ala and Val221Ala, can completely or mostly relieve feedback inhibition and maintain specific enzymatic activity [70][71][72].The feedback inhibition resistant mutants of CM/PD, CM/PDH and AS have also been identified [73][74][75].
Elimination of feedback inhibition has been shown to be key to microbial production of aromatic compounds (Fig. 4).For example, the combined utilization of DAHPS fbr and CM fbr in S. cerevisiae increased the Fig. 1 Schematic of cyanobacterial cell factories producing aromatic natural products.The properties, such as CO 2 fixation, abundant membrane, plentiful NADPH and ATP, and simple genetic manipulation, make cyanobacteria ideal candidates for the production of aromatic compounds flux to the AAAs synthesis pathway by a factor of 4.5 [76,77].The effect of this strategy on cyanobacteria is also remarkable.Direct expression of heterologous TAL enzymes in Synechococcus elongatus PCC 7942 (S. elongatus PCC 7942) resulted in low p-coumaric acid production (below 1.5 mg/L).Ni et al. increased the yield of precursor Tyr by overexpressing the feedback-inhibitorresistant DAHPS (DAHPS fbr ) derived from E. coli, which increased the yield of p-coumaric acid by more than 30 times [27].Releasing the feedback inhibition of DAHPS is the key to alleviate the production bottleneck of aromatic compounds, while in combination with other feedback-inhibition-resistant enzymes has additional effects.

Deshpande et al. individually and in combination overex-
pressed DAHPS fbr and AS fbr derived from E. coli to study their effect on the production of Trp in Synechocystis sp.PCC 6803.Overexpression of DAHPS fbr alone resulted in increased Trp titer, and overexpression of AS fbr alone had no significant effect on the yield.The engineered strain that overexpressed both enzymes achieved the highest Trp production, reaching 12.6 mg/L, with a growth rate similar to that of the wild type [37].An artificial feedback-inhibition-resistant cassette (FBR cassette) containing aroG fbr (encoding DAHPS fbr ) and pheA fbr (encoding CM/PD fbr ) was constructed in S. elongatus to improve the production of 2-phenylethanol (2-PE), the downstream  product of Phe.The FBR cassette increased the 2-PE yield by 48 times, increased the carbon fixation rate by more than 50%, and changed the carbon flux distribution, allocating more than 31.5% of the fixed carbon to the shikimate pathway [36].In addition, overexpression of shikimate kinase encoding gene aroK on the basis of the above metabolic modification to increase the flux into the shikimate pathway could further increase the production of 2-PE [40].The FBR cassette was recently used as a nonnative carbon sink to increase the cinnamic acid yield to 156.2 ± 4.1 mg/L, representing a 25-fold improvement compared with that of the parent strain [46].[38].
Eliminating feedback inhibition by using DAHPS fbr , AS fbr , CM/PD fbr , and CM/PDH fbr to increase carbon flux into the shikimate pathway is a general method for engineering microbial production of aromatic compounds.However, the regulation mechanism of AAAs synthesis in cyanobacteria is different from that in E. coli, and some modification methods might generate unexpected effects.In E. coli, overexpression of DAHPS and CM/ PDH led to the accumulation of more Tyr than Phe, but in cyanobacteria, with the same operation, more Phe was produced [78].In Synechocystis sp.PCC 6803, most of the prephanate was subsequently used to produce Phe rather than Tyr, and the activity of PheA is not negatively regulated by Phe [38].Detailed studies of the regulatory mechanisms of aromatic amino acids in different cyanobacteria species are still needed, which is important for developing appropriate strategies to remove feedback inhibition and accumulate precursors for the production of target products.

Blocking the competition pathway
The conversion of target products to byproducts and the diversion of key intermediates are important aspects affecting the yield (Fig. 4).Blocking the competition pathway by deleting the relevant enzyme is a possible solution.Xue et al. overexpressed TAL encoding gene sam8 from Saccharothrix espanaensis to produce p-coumaric acid in Synechocystis sp.PCC 6803, but obtained low levels of target products and more 4-vinylphenol, the decarboxylation product of p-coumaric acid.Subsequently, the protein encoded by slr1573 was identified as having strong laccase activity, capable of oxidizing a variety of phenolic substrates including p-coumaric acid.By knocking out slr1573 and overexpressing TAL enzyme, a 25-fold increase in p-coumaric acid titer was detected, and its downstream products were greatly reduced [32].Based on the elimination of feedback inhibition, Brey et al. further knocked out slr1573 gene and expressed XAL enzyme with both TAL and PAL activities, achieving 200.7 ± 30.1 mg/L p-coumaric acid and 114.1 ± 13.6 mg/L cinnamic acid [38].Arogenate is a Knocking out pathway genes is not always an appropriate strategy due to the potential for large metabolic burdens, alterations in genomic structure, or effects on other important metabolic processes.In the process of optimizing the production of p-coumaric acid and cinnamic acid in Synechocystis sp.PCC 6803, Kukil et al. reported that the engineered cyanobacteria lacking laccase exhibited slower growth, lower pigment content, and lower yield [42].Given the importance of AAAs synthesis for cyanobacteria survival, it may be useful to use reversibly inducible knockdown strategies to appropriately inhibit competition pathways.The expression of artificial sRNAs imposes little metabolic burden on host cells [79].Sun et al. introduced an artificial sRNA regulatory system into Synechocystis sp.PCC 6803, which increased the production of malonyl-CoA by 41% by interfering multiple genes necessary for fatty acid synthesis [80].Similar tools also achieved significant inhibition of target genes in the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 and the salt-tolerant marine cyanobacterium Synechococcus sp.PCC 7002 [81,82].A tunable gene repression system was developed by an inducible CRISPRi system to attenuate the expression of essential genes to varying degrees, producing 2-fold more lactate than the baseline engineered cell line [83].

Adaptive laboratory evolution
The regulatory mechanisms of cyanobacteria for the synthesis of aromatic compounds are quite complex and are variational across cyanobacteria species.Adaptive laboratory evolution, by applying suitable pressure to evolve chassis strains with high yields of aromatic compounds, has proven to be a universal and effective method (Fig. 4).ʟ-Phe was a potent inhibitor of DAHPS and PD, leading to 90% and 91% inhibition, respectively, at a concentration of 0.05 mM ʟ-Phe.Hall et al. cultured Synechocystis sp.29108 with the addition of 1 mM Phe and obtained several spontaneous mutants resistant to Phe, which excreted excess of Phe into the medium along with a smaller amount of Tyr.Most of the DAHPS mutants were completely desensitized to Phe inhibition, and possessed higher specific activities, ranging from 1.5 to 3.3 times greater than that of the wild type.The analysis of the Fig. 4 Schematic of strategies for regulating cyanobacteria metabolism and optimizing synthetic pathways.Engineered cyanobacteria cells that produce aromatic natural products can be constructed by eliminating regulatory mechanisms, blocking competing pathways, adaptive laboratory evolution, and optimizing the expression of pathway enzymes, as well as P450 enzymes mutants also provided in vivo evidence for the regulation of Tyr, that is, Tyr can activate PD activity [65].Labarre et al. demonstrated the existence of active transport systems operating on amino acids in Synechocystis sp.PCC 6803 by screening and analyzing spontaneous mutants resistant to toxic amino acids and analogs.Among them, mutants resistant to the above analogues were strong overproducers of Phe and Tyr [84].In another study, cyanobacteria Phormidium uncinatum was mutagenized by the mutagen N-methyl-N-nitro-N-nitrosoguanidine (NTG) and then screened for resistant mutants with several amino acid analogs.The mutants accumulated a variety of different amino acids, such as glutamic acid, Phe, and Tyr [85].
The tolerance of evolved strains to screening factors may come from the adjustment of other metabolic pathways.In-depth analysis of the genomic changes of the mutants, combined with other metabolic engineering strategies and optimization of culture technology will be conducive to further increases in yield.Deshpande et al. used DNA alkylating agent methyl-methanesulfonate (MMS) for random mutagenesis of Synechocystis sp.PCC 6803, followed by screening of mutants with Trp analogs.The mutant SYNY3-JV1 produced 17.2 ±1.5 mg/L of Trp, and its yield and growth rate were higher than those of the wild type.Sequencing results showed that all the Trpoverproducing mutants had fully segregated mutations in the CM-encoding gene aroH, providing novel mutations in CM that enhance Trp accumulation.Further overexpression of the feedback-resistant enzymes DAHPS fbr and AS fbr in the mutant increased the Trp yield to 46.1 mg/L, and even produced 212 ± 23 mg/L Trp at elevated CO 2 levels and higher inoculation densities [37].Kukil et al. obtained laboratory-evolved mutants of Synechocystis sp.PCC 6803 by applying selective pressure from Phe.The Phe-resistant mutants secreted Phe and Tyr into the medium and acquired mutations in the same regulatory domain of DAHPS.One of the mutants, PRM8, produced 610 ± 196 mg/L Phe in a high-density cultivation (HDC) system.It is worth noting that allosteric deregulation caused by evolution may also affect the activity of related enzymes, resulting in a decrease in the production of target chemicals.The exogenous enzymes PAL or TAL were overexpressed in ten mutants to test the production of cinnamic acid and p-coumaric acid.During the culture in shake flasks, the yield of both products of almost all mutants was lower than that of their respective wild type control strains.During the high-density cultivation, only one mutant, PRM8, had a better yield than the control strain, producing 1108.8± 150 mg/L p-coumaric acid and 1116.7 ± 314 mg/L cinnamic acid, respectively [45].
The frequency of mutation is a key parameter for obtaining evolutionary strains.A variety of synthetic tools have been developed in other species to increase the mutation frequency of strains.Activation-induced cytosine deaminase (AID) converts cytosine (C) from single-stranded DNA in the R-loop to uracil (U) by deamination, thereby effecting a C to thymine (T) substitution [86].Wang et al. fused the DNA helicase DnaB to AID to form a helicase-AID enzyme complex, aiming to randomly introduce base substitutions throughout the chromosome in E. coli.Compared with the wild type, DnaB-AID increased the mutagenesis frequency by 2.5 × 10 3 times, and achieved a 371.4% increase in β-carotene production following four rounds of editing.The designed Helicase-AID complex in S. cerevisiae, MCM5-AID, made the average editing efficiency 2.1 ± 0.4 × 10 3 times greater than the native genomic mutation rate, and improved β-carotene production by 75.4% following eight rounds of editing [87].By fusing an unspecific single-stranded DNA-binding protein to a cytidine deaminase, Pan et al. established a random base editing (rBE) system capable of introducing C into T mutations in a genome-wide manner to facilitate the evolution of S. cerevisiae [88].Adjusting and applying these synthetic components and engineering strategies to cyanobacteria, in combination with specific screening methods, may help to obtain ideal photosynthetic chassis cells for the production of natural compounds, including aromatic products.

Regulation of heterologous enzyme expression
Aromatic natural products are mostly secondary metabolites of plants, not naturally synthesized by cyanobacteria.When heterologous genes are introduced, some rare codons may not be recognized by the host's transcription/translation mechanisms, resulting in inefficient expression [89].The expression levels of pathway enzymes is certainly increased after codon optimization [90].Xue et al. optimized the C3H encoding gene ref8 from Arabidopsis thaliana by replacing the more frequently used codon and adjusting the AT/GC ratio, and heterogeneously expressed it in Synechocystis sp.PCC 6803 to produce caffeic acid.After codon optimization, the protein product reached a relatively high level, and the yield of caffeic acid increased from 5 to 7.2 mg/L [31].
Testing enzymes from different sources and adjusting their expression levels have been shown to be important for the synthesis of specific aromatic compounds.In S. elongatus PCC 7942, TAL from Saccharothrix espanaensis was more efficient than that from Rhodobacter sphaeroides in producing p-coumaric acid, C3H from Arabidopsis thaliana had better enzymatic kinetics than that from Saccharothrix espanaensis in producing caffeic acid, and ADH from Synechocystis sp.PCC 6803 was more efficient than that from E. coli in producing 2-PE [27,36].Ni et al. constructed two ways to produce 2-PE in S. elongatus PCC 7942, the phenylacetaldehyde synthase (PAAS)-mediated route and the 2-keto acid decarboxylase (KDC)-mediated route.Initially, the yield of the former route is higher than that of the latter.However, after removing feedback inhibition, the latter produced 1.46 times more than the former [36].Kukil and Lindberg tested five PAL enzymes from different species to produce cinnamic acid in Synechocystis sp.PCC 6803, indicating the PAL from Arabidopsis thaliana with the highest specific production [42].
Precise regulation of the expression intensity of pathway enzymes by regulatory elements such as promoters, RBS and ribose switches can improve the production of compounds [91].The constitutive promoters commonly used in cyanobacteria are Pcpc, PpsbA, Prbc, Ptrc, and J23-series promoters from the iGEM Registry of Standard Biological Parts [92].The yield of target products is related to the selection of suitable promoters and RBS to drive the full expression of pathway enzymes.For example, the Synechocystis sp.PCC 6803 expressing TAL from Rhodobacter sphaeroides under Ptrc1O promoter produced p-coumaric acid at a rate three times higher than that under Ptrc1Ocore promoter [35].The super-strong promoter Pcpc560, which contains multiple transcription factor binding sites, was shown to be able to produce at a level of up to 15% of the total soluble protein in Synechocystis sp.PCC 6803 [93].Gao et al. used promoter Pcpc560 to express PAL and C4H in Synechocystis sp.PCC 6803, achieving 131 mg/L of p-coumaric acid after 7 days of cultivation [39].However, excess heterologous proteins may not lead to higher titers, but instead result in metabolic burden and affect cell growth [42].Exploring the optimal expression intensity of pathway enzymes via an inducible system is an effective way to increase the yield of aromatic compounds.Ni et al. induced with different concentrations of IPTG to assess the effect of gene transcription levels on p-coumaric acid production, obtaining the highest product titer of 128.2 mg/L at 0.5 mM IPTG.At the same time, the authors also pointed out that the regulation of IPTG induction system has leakage expression, which is consistent with other studies [27,94].Subsequently, tightly regulated induction systems with low leakage and a large dynamic range can be attempted to finely regulate the pathway enzymes of aromatic natural products, such as rhamnose-induced and RhaS-regulated promoter P rhaBAD , arabinose-induced and AraC-regulated promoter P BAD , metal-responsive promoters P nrsB , and modified theophylline-dependent riboswitch F [95][96][97][98].
In the process of de novo synthesis of natural products, the low utilization rate of intermediate products will lead to low yields and affect cell growth.Co-locating pathway enzymes to form multi-enzyme complexes could increase the concentration of local metabolites and thus improve the conversion efficiency of intermediate products (Fig. 4) [99].Ni et al. coupled 4CL to different type III PKS genes by amino-acid linker to improve the conversion of p-coumaroyl-CoA, obtained 2.2 mg/L resveratrol, 4.3 mg/L naringenin, and 2.9 mg/L bisdemethoxycurcumin in cyanobacteria, respectively, which were 2.1 times, 1 time and 1.2 times higher than those expressing separate pathway enzymes [27].Li et al. explored an automated cell-free high-throughput workflow to screen the optimal linker for the fusion of 4CL and CUS, creating fusion proteins with better spatial proximity, resulting in a 71% improvement in the curcumin titer [46].Synthesized scaffolds provide a co-localization method to flexibly regulate metabolic pathways catalyzed by multiple enzymes.Dueber et al. used SH3, PDZ, and GBD domains as interaction modules to construct synthetic protein scaffolds for mevalonate production in E. coli.By optimizing synthetic scaffold expression levels to balance the scaffold to enzyme ratio and the stoichiometries of enzymes, achieving 77-fold improvement in product titer [100].Wang and Yu constructed the scaffolds to recruit the 4CL and STS of resveratrol biosynthesis pathway and optimized the number of domain repeats in the scaffolds, with a yield 2.7 times higher than that with fusion proteins in yeast cells [101].Although the optimal synthetic scaffold architecture for different synthetic pathways needs to be explored, this method has shown great potential for improving the production of natural compounds [102].

Functional expression of cytochrome P450 enzymes
Cytochrome P450 enzymes (P450s) are capable of catalyzing the reaction of hydroxylation, aryl and phenolic ring-coupling, double bond epoxidation, C-C bond cleavage, and oxidative rearrangement of carbon skeleton [103].P450s, such as C4H, C3H, flavonoid hydroxylase, and flavonoid synthase, contribute to the production and diversity of aromatic natural products [104].Plant P450s are membrane-bound, which are difficult to express in prokaryotic systems such as E. coli.Moreover, most P450s require REDOX chaperones to transfer two electrons of NADPH to the reaction center to activate oxygen molecules, but the native NADPH regeneration metabolism of bacteria and yeast is generally insufficient to support high levels of utilization [27].Cyanobacteria contain a well-developed intracellular membrane system thylakoid, as well as abundant NADPH and ATP produced by the photosynthetic electron transport chain, allowing P450s to assemble and function properly (Fig. 4).
Cyanobacteria use photosystem II (PSII) and photosystem I (PSI) complexes on thylakoids to capture light energy, drive electron transport, and release oxygen.Electrons are extracted from water by PSII and delivered to PSI via electron transport chain.PSI utilizes light energy to excite electrons and transfers them to electron transfer protein ferredoxin (Fd), which in turn reduces ferredoxin-NADP + reductase (FNR) to further catalyze the formation of NADPH [105][106][107].Coupling P450s with PSI allows electrons extracted from the photosynthetic electron transport chain to directly drive the enzymatic reaction [108].Lassen et al. linked the catalytic domain of P450 CYP79A1 from Sorghum bicolor to the PSI subunit PsaM in Synechococcus sp.PCC 7002, obtaining PsaM-CYP79A1 fusion protein.
The fusion protein was shown to co-locate in the thylakoids and receive electrons from PSI via Fd, while maintaining CYP79A1 enzymatic activity to convert the substrate Tyr to p-hydroxyphenylacetaldoxime [33].By taking advantage of Fd as a direct electron transfer protein rather than an NADPH-dependent P450 oxidoreductase, the catalytic efficiency and turnover rate of P450s were improved [109].To divert more photosynthetic reducing power to engineered metabolism, the fusions that covalently connect Fd to P450 CYP79A1 were designed and targeted to the chloroplast in Nicotiana benthamiana by Mellor et al.One of the P450-Fd fusions could obtain electrons directly from PSI via fused Fd, without the need for a dedicated reductase, showing stronger competitiveness with endogenous electron sinks and higher in vivo activity than the native enzyme [110].Wlodarczyk et al. engineered Synechocystis sp.PCC 6803 to express a complete dhurrin pathway composed of membrane bound P450s and a soluble glycosyltransferase, producing 66 mg/L p-hydroxyphenylacetaldoxime and 5 mg/L dhurrin in lab-scale cultures.The research reported an encouraging demonstration that eukaryotic P450s could insert into the thylakoid membrane of cyanobacteria in the absence of N-terminal targeting signals and receive electrons delivered by PSI and Fd [34].
In addition, the diversity of natural P450s derived from cyanobacteria species is high, with some unusual catalytic diversity and resemblance to eukaryotic P450s [111].For instance, CYP110E1 from the Nostoc sp.strain PCC 7120 was found to be a kind of flavone synthase, being able to hydroxylate naringenin and (hydroxyl) flavanones into apigenin and (hydroxyl) flavones [112].Enhancing the conversion efficiency of exogenous P450s by enzyme engineering and exploiting abundant endogenous P450s are the cornerstones for the production of high value complex aromatic natural products, especially flavonoids.

Culture process development
Cyanobacteria usually exhibit lower cell density and slower reaction rate in non-optimal photobioreactors.Production processes based on the cyanobacteria cell factories are limited by low space-time yields as a result of slow, light-dependent growth [113].The optimization of external conditions, such as cultivation medium, light availability, and gas supply, is an available solution to for enhancing the production (Fig. 5).Light is a limiting factor affecting the growth of cyanobacteria [114].Constant light and moderately higher frequency of light fluctuations were shown to be beneficial for biomass growth [115,116].Carbon availability may be another challenge for the growth of cyanobacteria, as 0.04% CO 2 in ambient air is not enough for optimal biomass growth.Continuous aeration with CO 2 -enriched air (5%, v/v) promoted the growth of engineered S. elongatus PCC 7942 and increased the production rate of p-coumaric acid to 10.1 mg/L/day, which was 1.8 times higher than that of the resting culture without continuous aeration [27].In another study of p-coumaric acid production, pumping CO 2 into the engineered Synechocystis sp.PCC 6803 culture increased the growth rate by 2-folds [39].It is important to note that the pH of the medium should be closely monitored when supplementing with CO 2 -enriched gases.The use of sodium bicarbonate as a carbon source could replenish the substrate and also buffer the medium pH [117].Cell growth and chemical production could be improved by adjusting the medium through co-substrate feeding strategy [118,119].Usai et al. tested several metabolite doping conditions to improve 2-PE production and found that doping with 0.3 g/L Phe was effective, which might relieve the competition between 2-PE synthetic pathway and endogenous routes for the common precursor Phe.By combining two strategies of metabolite doping and metabolic engineering, the engineered strain obtained 285 ± 15 mg/L of 2-PE, about 2.4 times higher than that of the strain constructed by Ni et al [40].
Recently, a small-scale high-density cultivation (HDC) setup for rapid growth of cyanobacteria to ultra-high cell density was developed, which consisted of a two-tier vessel, using membrane-mediated CO 2 supply, in combination with rapid turbulent mixing, high photon flux density, and optimized layer thickness of the culture [120,121].Kukil et al. tested engineered Synechocystis sp.PCC 6803 under the HDC system, and observed that the total yields of cinnamic acid and p-coumaric acid significantly increased to 797.8 ± 153.3 mg/L and 411.6 ± 94.9 mg/L, respectively, while the productivity of single cells did not increase compared with that under standard dilution conditions [42].Some studies of culture tests in HDC devices showed that the optimal culture conditions could improve the productivity of cyanobacteria and even make it close to the productivity of heterotrophic host [121,122].
The design of bioprocesses is an important link for promoting sustainable development and circular economy, which can realize rational utilization of waste resources to reduce the fermentation cost of engineering cyanobacteria and increase the production scale.Usai et al. evaluated the effects of four dairy effluents on cyanobacteria growth and showed that both tank-washing water and the liquid effluent of exhausted sludge were suitable nutrient sources [41].In response to this, the researchers designed a biological fermentation process using nutrient-rich wastewater, and engineered cyanobacteria were able to generate 205 mg/L 2-PE, avoiding the use of fresh water resources and achieving sustainable production of 2-PE in a low-cost and environmentally friendly manner [41].Currently, multiple photobioreactors (PBRs) for largescale fermentation have been developed, such as column PBR and flat panel PBR, which can improve light supply and increase productivity and biomass [123,124].Selecting a suitable culture system according to the characteristics of the chassis cells and bioprocess will further expand the production scale of engineered cyanobacteria.

Production by microbial cooperation
Synthetic phototrophic microbial consortia allow the division of labor of different species to express specific designed metabolic modules, thereby achieving the efficient light-driven synthesis of CO 2 into broad-spectrum target products [125].Typically, modified cyanobacteria are used as carbon sequestration modules to convert CO 2 into carbohydrates or stable intermediates and secrete them into medium, while engineered heterotrophs are used as biosynthesis modules to produce target products [126,127].In the highly compatible phototrophic community developed by Li et al., the engineered S. elongatus containing sucrose permease encoding gene cscB produced and secreted sucrose as a CO 2 sequestration module, the Vibrio natriegens with high growth rate and strong sucrose utilization capacity acted as super-coupled module to efficiently convert sucrose into different chemicals (Fig. 6a).By introducing specific synthetic genes into the coupled module, the symbiotic system produced 24.7 mg/L p-coumaric acid [44].
There are several limitations in the production of several aromatic natural products by photosynthetic microorganisms, such as the decomposition of photosensitive products (e.g., curcumin), the escape of volatile products (e.g., styrene), and the undesirable conversion of products (e.g., aromatic aldehydes) mediated by endogenous enzymes [46,125,128].In response to these challenges, Li et al. developed a novel strategy of integration of photosynthesis and resting cellular catalysis (iPRCC) (Fig. 6b).The carbon sequestration module was constructed based on engineered S. elongatus PCC 7942, to convert CO 2 into stable mediator chemicals, including cinnamic acid and its derived carboxylic acids.Based on E. coli and S. elongatus, multiple cellular catalysis modules were constructed through multiple gene editing, pathway enzyme overexpression and high-throughput workflows to convert the above intermediates into target compounds.By interfacing the carbon sequestration module with different resting cellular catalysis modules, CO 2 was converted directly into volatile styrene, photosensitive products (curcumin and scopolamine), intracellular unstable products (cinnamaldehyde and vanillin), resveratrol and naringin, with yields increased by tens to hundreds of times compared to those of monoculture.Through large-scale fermentation, the yield of most products reaches a level of gram-per-liter, is even comparable to that of engineered heterotrophs [46].The plug-and-play iPRCC strategy is also applicable to the synthesis of other types of products, expanding the scope of light-driven biosynthesis and demonstrating the potential for large-scale sustainable production.

Conclusions
Cyanobacteria have proven to be one of the ideal candidates for the production of aromatic natural products, and this carbon-neutral production mode has unique advantages in the context of climate change.However, the variety of products currently produced by cyanobacteria cell factories is relatively concentrated and is usually a proof of concept.Therefore, further work is needed to explore the synthesis of more diverse Fig. 6 Schematic of microbial cooperation for the production of aromatic natural products.a Cyanobacteria convert CO 2 into sucrose and secrete it into the culture medium.The heterotroph acts as a collaborator to use sucrose as carbon source to grow and produce specific aromatic compounds, while producing CO 2 to promote the growth of cyanobacteria.b The integration of photosynthesis and resting cellular catalysis (iPRCC) strategy.The iPRCC strategy is divided into carbon sequestration module and cellular catalysis module.Unlike phototrophic community, the partners of this strategy do not cooperate in the form of co-culture.Cyanobacteria act as carbon sequestration modules, converting CO 2 into stable intermediate compounds and secreting them out of the cell.The multiple cellular catalysis modules convert the above intermediates into different aromatic compounds aromatic natural products and to increase the production scale and yield of specific products.In addition to testing multiple plant-derived synthases, it is also possible to focus on the rich genomic and transcriptome information of cyanobacteria to explore potential synthetic pathways for a variety of aromatic natural products.Haematococcus pluvialis was proven to convert externally fed phenylpropanoid precursors such as ferulic acid to a range of vanilla flavor metabolites [129].Dalponte et al. identified and characterized an AcyF prenyltransferase from the cyanobactin gene cluster of Anabaena sp.UHCC-0232, which can catalyze the prenylation of the indole N-1 position of Trp to synthesize tryptophan derivatives [130].Recently, a new database CyanoOmicsDB was reported [131], which can provide comprehensive functional information for cyanobacterial gene, such as predicted gene function, amino acid sequences, homologs, etc.
In order to improve the production efficiency of cyanobacteria for aromatic compounds, it is necessary to jointly solve the problems of pathway regulation, carbon fixation and carbon allocation, and REDOX balance.As described in many of the above works, the realization of efficient cyanobacteria cell factories requires a combination of strategies.However, these measures often impose an unexpected metabolic burden on the host cell, which in turn affects production performance [132].Metabolic flux analysis quantitatively determines the intracellular flows of carbon within complex biochemical networks, and provides valuable information on the analysis of regulatory bottlenecks, diverging pathways at branch point, discovering unusual pathways, and identifying the potential ways to improve production performance [133,134].Systematically balancing the metabolic networks of host strains through metabolic flux analysis and multiomics information is important for engineering strains to maximize the production of target products [133,135,136].
Overall, cyanobacteria cell factory has opened up new possibilities for the sustainable production of aromatic natural products in a carbon-neutral way.It is believed that continued and innovative development will allow cyanobacteria to produce more complex, diverse and valuable aromatic natural products on a larger scale.We hope that this review provides some refined and useful information for further research in this area.

Fig. 2
Fig. 2 The chemical structure of aromatic natural products produced by engineered cyanobacteria

Fig. 5
Fig. 5 Schematic of cyanobacteria culture process development.Light intensity, CO 2 concentration, medium and culture apparatus are important factors affecting the fermentation of cyanobacteria

Table 1
Aromatic natural products produced by engineered cyanobacteria