Comparing in planta accumulation with microbial routes to set targets for a cost-competitive bioeconomy.

Plants and microbes share common metabolic pathways for producing a range of bioproducts that are potentially foundational to the future bioeconomy. However, in planta accumulation and microbial production of bioproducts have never been systematically compared on an economic basis to identify optimal routes of production. A detailed technoeconomic analysis of four exemplar compounds (4-hydroxybenzoic acid [4-HBA], catechol, muconic acid, and 2-pyrone-4,6-dicarboxylic acid [PDC]) is conducted with the highest reported yields and accumulation rates to identify economically advantaged platforms and breakeven targets for plants and microbes. The results indicate that in planta mass accumulation ranging from 0.1 to 0.3 dry weight % (dwt%) can achieve costs comparable to microbial routes operating at 40 to 55% of maximum theoretical yields. These yields and accumulation rates are sufficient to be cost competitive if the products are sold at market prices consistent with specialty chemicals ($20 to $50/kg). Prices consistent with commodity chemicals will require an order-of-magnitude-greater accumulation rate for plants and/or yields nearing theoretical maxima for microbial production platforms. This comparative analysis revealed that the demonstrated accumulation rates of 4-HBA (3.2 dwt%) and PDC (3.0 dwt%) in engineered plants vastly outperform microbial routes, even if microbial platforms were to reach theoretical maximum yields. Their recovery and sale as part of a lignocellulosic biorefinery could enable biofuel prices to be competitive with petroleum. Muconic acid and catechol, in contrast, are currently more attractive when produced microbially using a sugar feedstock. Ultimately, both platforms can play an important role in replacing fossil-derived products.

Selective microbial production of lacto-N-fucopentaose I in Escherichia coli using engineered α-1,2-fucosyltransferases.

Lacto-N-fucopentaose I (LNFP I) is the second most abundant fucosylated human milk oligosaccharide (HMO) in breast milk after 2'-fucosyllactose (2'-FL). Studies have reported that LNFP I exhibits antimicrobial activity against group B Streptococcus and antiviral effects against Enterovirus and Norovirus. Microbial production of HMOs by engineered Escherichia coli is an attractive, low-cost process, but few studies have investigated production of long-chain HMOs, including the pentasaccharide LNFP I. LNFP I is synthesized by α1,2-fucosyltransfer reaction to the N-acetylglucosamine moiety of the lacto-N-tetraose skeleton, which is catalyzed by α1,2-fucosyltransferase (α1,2-FucT). However, α1,2-FucTs competitively transfer fucose to lactose, resulting in formation of the byproduct 2'-FL. In this study, we constructed LNFP I-producing strains of E. coli with various α1,2-fucTs, and observed undesired 2'-FL accumulation during fed-batch fermentation, although, in test tube assays, some strains produced LNFP I without 2'-FL. We hypothesized that promiscuous substrate selectivity of α1,2-FucT was responsible for 2'-FL production. Therefore, to decrease the formation of byproduct 2'-FL, we designed 15 variants of FsFucT from Francisella sp. FSC1006 by rational and semi-rational design approaches. Five of these variants of FsFucT surpassed a twofold reduction in 2'-FL production compared with wild-type FsFucT while maintaining comparable levels of LNFP I production. These designs encompassed substitutions in either a loop region of the enzyme (residues 154-171), or in specific residues (Q7, H162, and L164) that influence substrate binding either directly or indirectly. In particular, the E. coli strain that expressed FsFucT_S3 variants, with a substituted loop region (residues 154-171) forming an α-helix structure, achieved an accumulation of 19.6 g/L of LNFP I and 0.04 g/L of 2'-FL, while the E. coli strain expressing the wild-type FsFucT accumulated 12.2 g/L of LNFP I and 5.85 g/L of 2'-FL during Fed-bach fermentation. Therefore, we have successfully demonstrated the selective and efficient production of the pentasaccharide LNFP I without the byproduct 2'-FL by combining protein engineering of α1,2-FucT designed through in silico structural modeling of an α1,2-FucT and docking simulation with various ligands, with metabolic engineering of the host cell.

Advances in microbial production of geraniol: from metabolic engineering to potential industrial applications.

Geraniol, an acyclic monoterpene alcohol, has significant potential applications in various fields, including: food, cosmetics, biofuels, and pharmaceuticals. However, the current sources of geraniol mainly include plant tissue extraction or chemical synthesis, which are unsustainable and suffer severely from high energy consumption and severe environmental problems. The process of microbial production of geraniol has recently undergone vigorous development. Particularly, the sustainable construction of recombinant Escherichia coli (13.2 g/L) and Saccharomyces cerevisiae (5.5 g/L) laid a solid foundation for the microbial production of geraniol. In this review, recent advances in the development of geraniol-producing strains, including: metabolic pathway construction, key enzyme improvement, genetic modification strategies, and cytotoxicity alleviation, are critically summarized. Furthermore, the key challenges in scaling up geraniol production and future perspectives for the development of robust geraniol-producing strains are suggested. This review provides theoretical guidance for the industrial production of geraniol using microbial cell factories.

Fermentative aminopyrrolnitrin production by metabolically engineered Corynebacterium glutamicum.

Aminopyrrolnitrin (APRN), a natural halogenated phenylpyrrole derivative (HPD), has strong antifungal and antiparasitic activities. Additionally, it showed 2.8-fold increased photostability compared to pyrrolnitrin, a commercially available HPD with antimicrobial activity. For microbial production of APRN, we first engineered anthranilate phosphoribosyltransferase encoded by trpD from Corynebacterium glutamicum, resulting in a TrpDA162D mutation that exhibits feedback-resistant against L-tryptophan and higher substrate affinity compared to wild-type TrpD. Plasmid-borne expression of trpDA162D in C. glutamicum TP851 strain with two copies of trpDA162D in the genome led to the production of 3.1 g/L L-tryptophan in flask culture. Subsequent step for L-tryptophan chlorination into 7-chloro-L-tryptophan was achieved by introducing diverse sources of genes encoding tryptophan 7-halogenase (PrnA or RebH) and flavin reductase (Fre, PrnF, or RebF). The combined expression of prnA from Serratia grimesii or Serratia plymuthica with flavin reductase gene from Escherichia coli, Pseudomonas fluorescens, or Lechevalieria aerocolonigenes yielded higher production of 7-chloro-L-tryptophan in comparison to other sets of two-component systems. In the next step, production of putative monodechloroaminopyrrolnitrin (MDAP) from 7-chloro-L-tryptophan was achieved through the expression of prnB encoding MDAP synthase from S. plymuthica or P. fluorescens. Finally, an artificial APRN biosynthetic pathway was constructed by simultaneously expressing genes coding for tryptophan 7-halogenase, flavin reductase, MDAP synthase, and MDAP halogenase (PrnC) from different microbial sources within the L-tryptophan-producing TP851 strain. As prnC from S. grimesii or S. plymuthica was introduced into the host strain, which carried plasmids expressing prnA from S. plymuthica, fre from E. coli, and prnB from S. plymuthica, APN3639 and APN3638 accumulated 29.5 mg/L and 28.1 mg/L of APRN in the culture broth. This study represents the first report on the fermentative APRN production by metabolically engineered C. glutamicum.

Engineered biosynthesis of plant heteroyohimbine and corynantheine alkaloids in Saccharomyces cerevisiae.

Monoterpene indole alkaloids (MIAs) are a class of natural products comprised of thousands of structurally unique bioactive compounds with significant therapeutic values. Due to difficulties associated with isolation from native plant species and organic synthesis of these structurally complex molecules, microbial production of MIAs using engineered hosts are highly desired. In this work, we report the engineering of fully integrated Saccharomyces cerevisiae strains that allow de novo access to strictosidine, the universal precursor to thousands of MIAs at 30-40 mg/L. The optimization efforts were based on a previously reported yeast strain that is engineered to produce high titers of the monoterpene precursor geraniol through compartmentalization of mevalonate pathway in the mitochondria. Our approaches here included the use of CRISPR-dCas9 interference to identify mitochondria diphosphate transporters that negatively impact the titer of the monoterpene, followed by genetic inactivation; the overexpression of transcriptional regulators that increase cellular respiration and mitochondria biogenesis. Strain construction included the strategic integration of genes encoding both MIA biosynthetic and accessory enzymes into the genome under a variety of constitutive and inducible promoters. Following successful de novo production of strictosidine, complex alkaloids belonging to heteroyohimbine and corynantheine families were reconstituted in the host with introduction of additional downstream enzymes. We demonstrate that the serpentine/alstonine pair can be produced at ∼5 mg/L titer, while corynantheidine, the precursor to mitragynine can be produced at ∼1 mg/L titer. Feeding of halogenated tryptamine led to the biosynthesis of analogs of alkaloids in both families. Collectively, our yeast strain represents an excellent starting point to further engineer biosynthetic bottlenecks in this pathway and to access additional MIAs and analogs through microbial fermentation. ONE SENTENCE SUMMARY: An Saccharomyces cerevisiae-based microbial platform was developed for the biosynthesis of monoterpene indole alkaloids, including the universal precursor strictosidine and further modified heteroyohimbine and corynantheidine alkaloids.

High-throughput genetic engineering tools for regulating gene expression in a microbial cell factory.

With the rapid advances in biotechnological tools and strategies, microbial cell factory-constructing strategies have been established for the production of value-added compounds. However, optimizing the tradeoff between the biomass, yield, and titer remains a challenge in microbial production. Gene regulation is necessary to optimize and control metabolic fluxes in microorganisms for high-production performance. Various high-throughput genetic engineering tools have been developed for achieving rational gene regulation and genetic perturbation, diversifying the cellular phenotype and enhancing bioproduction performance. In this paper, we review the current high-throughput genetic engineering tools for gene regulation. In particular, technological approaches used in a diverse range of genetic tools for constructing microbial cell factories are introduced, and representative applications of these tools are presented. Finally, the prospects for high-throughput genetic engineering tools for gene regulation are discussed.

Rational design of GDP­D­mannose mannosyl hydrolase for microbial L­fucose production.

BACKGROUND: L­Fucose is a rare sugar that has beneficial biological activities, and its industrial production is mainly achieved with brown algae through acidic/enzymatic fucoidan hydrolysis and a cumbersome purification process. Fucoidan is synthesized through the condensation of a key substance, guanosine 5'­diphosphate (GDP)­L­fucose. Therefore, a more direct approach for biomanufacturing L­fucose could be the enzymatic degradation of GDP­L­fucose. However, no native enzyme is known to efficiently catalyze this reaction. Therefore, it would be a feasible solution to engineering an enzyme with similar function to hydrolyze GDP­L­fucose. RESULTS: Herein, we constructed a de novo L­fucose synthetic route in Bacillus subtilis by introducing heterologous GDP­L­fucose synthesis pathway and engineering GDP­mannose mannosyl hydrolase (WcaH). WcaH displays a high binding affinity but low catalytic activity for GDP­L­fucose, therefore, a substrate simulation­based structural analysis of the catalytic center was employed for the rational design and mutagenesis of selected positions on WcaH to enhance its GDP­L­fucose­splitting efficiency. Enzyme mutants were evaluated in vivo by inserting them into an artificial metabolic pathway that enabled B. subtilis to yield L­fucose. WcaHR36Y/N38R was found to produce 1.6 g/L L­fucose during shake­flask growth, which was 67.3% higher than that achieved by wild­type WcaH. The accumulated L­fucose concentration in a 5 L bioreactor reached 6.4 g/L. CONCLUSIONS: In this study, we established a novel microbial engineering platform for the fermentation production of L­fucose. Additionally, we found an efficient GDP­mannose mannosyl hydrolase mutant for L­fucose biosynthesis that directly hydrolyzes GDP­L­fucose. The engineered strain system established in this study is expected to provide new solutions for L­fucose or its high value­added derivatives production.

Microbial Hyaluronic Acid Production: A Review.

Microbial production of hyaluronic acid (HA) is an area of research that has been gaining attention in recent years due to the increasing demand for this biopolymer for several industrial applications. Hyaluronic acid is a linear, non-sulfated glycosaminoglycan that is widely distributed in nature and is mainly composed of repeating units of N-acetylglucosamine and glucuronic acid. It has a wide and unique range of properties such as viscoelasticity, lubrication, and hydration, which makes it an attractive material for several industrial applications such as cosmetics, pharmaceuticals, and medical devices. This review presents and discusses the available fermentation strategies to produce hyaluronic acid.

Recent advances in the production of malic acid by native fungi and engineered microbes.

Malic acid is mainly produced by chemical methods which lead to various environmental sustainability concerns associated with CO2 emissions and resulting global warming. Since malic acid is naturally synthesized, microorganisms offer an eco-friendly and cost-effective alternative for its production. An additional advantage of microbial production is the synthesis of pure L-form of malic acid. Due to its numerous applications, biotechnologically- produced L-malic acid is a much sought-after platform chemical. Malic acid can be produced by microbial fermentation via oxidative/reductive TCA and glyoxylate pathways. This article elaborates the potential and limitations of high malic acid producing native fungi belonging to Aspergillus, Penicillium, Ustilago and Aureobasidium spp. The utilization of industrial side streams and low value renewable substrates such as crude glycerol and lignocellulosic biomass is also discussed with a view to develop a competitive bio-based production process. The major impediments present in the form of toxic compounds from lignocellulosic residues or synthesized during fermentation along with their remedial measures are also described. The article also focuses on production of polymalic acid from renewable substrates which opens up a cost-cutting dimension in production of this biodegradable polymer. Finally, the recent strategies being employed for its production in recombinant organisms have also been covered.

Dual ß-oxidation pathway and transcription factor engineering for methyl ketones production in Saccharomyces cerevisiae.

Methyl ketones (MK) are highly valuable fatty acid derivatives with broad applications. Microbes based biosynthesis represents an alternative route for production of these usually fossil based chemicals. In this study, we reported metabolic engineering of Saccharomyces cerevisiae to produce MK, including 2-nonanone, 2-undecanone, 2-tridecanone and 2-pentadecanone. Besides enhancing inherent peroxisomal fatty acids ß-oxidation cycle, a novel heterologous cytosolic fatty acids ß-oxidation pathway was constructed, and this resulted in an increased production of MK by 2-fold. To increase carbon fluxes to methyl ketones, the supply of precursors was enhanced by engineering lipid metabolism, including improving the intracellular biosynthesis of acyl-CoAs, weakening the consumption of acyl-CoAs for lipids storage, and reinforcing activation of free fatty acids to acyl-CoAs. Hereby the titer of MK was improved by 7-fold, reaching 143.72 mg/L. Finally, transcription factor engineering was employed to increase the biosynthesis of methyl ketones and it was found that overexpression of ADR1 can mimic the oleate activated biogenesis and proliferation of peroxisomes, which resulted in a further increased production of MK by 28%. With these modifications and optimization, up to 845 mg/L total MK were produced from glucose in fed-batch fermentation, which is the highest titer of methyl ketones reported produced by fungi.

Bioproduction of l- and d-lactic acids: advances and trends in microbial strain application and engineering.

Lactic acid is an important platform chemical used in the food, agriculture, cosmetic, pharmaceutical, and chemical industries. It serves as a building block for the production of polylactic acid (PLA), a biodegradable polymer, which can replace traditional petroleum-based plastics and help to reduce environmental pollution. Cost-effective production of optically pure l- and d-lactic acids is necessary to achieve a quality and thermostable PLA product. This paper evaluates research advances in the bioproduction of l- and d-lactic acids using microbial fermentation. Special emphasis is given to the development of metabolically engineered microbial strains and processes tailored to alternative and flexible feedstock concepts such as: lignocellulose, glycerol, C1-gases, and agricultural-food industry byproducts. Alternative fermentation concepts that can improve lactic acid production are discussed. The potential use of inducible gene expression systems for the development of biosensors to facilitate the screening and engineering of lactic acid-producing microorganisms is discussed.

Metabolic engineering of Yarrowia lipolytica for terpenoids production: advances and perspectives.

Terpenoids are a large family of natural products with diversified structures and functions that are widely used in the food, pharmaceutical, cosmetic, and agricultural fields. However, the traditional methods of terpenoids production such as plant extraction and chemical synthesis are inefficient due to the complex processes, high energy consumption, and low yields. With progress in metabolic engineering and synthetic biology, microbial cell factories provide an interesting alternative for the sustainable production of terpenoids. The non-conventional yeast, Yarrowia lipolytica, is a promising host for terpenoid biosynthesis due to its inherent mevalonate pathway, high fluxes of acetyl-CoA and NADPH, and the naturally hydrophobic microenvironment. In this review, we highlight progress in the engineering of Y. lipolytica as terpenoid biomanufacturing factories, describing the different terpenoid biosynthetic pathways and summarizing various metabolic engineering strategies, including progress in genetic manipulation, dynamic regulation, organelle engineering, and terpene synthase variants.

Microbial xylitol production.

Xylitol is pentahydroxy sugar alcohol, existing in very trace amount in fruits and vegetables, and finds varied application in industries like food, pharmaceuticals, confectionaries, etc. and is of prime importance to health. Owing to its trace occurrence in nature and considerable increase in market demand that exceeds availability, alternate production through biotechnological and chemical approach is in process. Biochemical production involves substrates like lignocellulosic biomasses and industrial effluents and is an eco-friendly process with high dependency on physico-chemical parameters. Although the chemical processes are faster, high yielding and economical, they have a great limitation as usage of toxic chemicals and thus need to be regulated and replaced by an environment friendly approach. Microbes play a major role in xylitol production through a biotechnological process towards the development of a sustainable system. Major microbes working on assimilation of xylose for production of xylitol include Candida tropicalis, Candida maltose, Bacillus subtilis, Debaromyces hansenii, etc. The present review reports all probable microbial xylitol production biochemical pathways encompassing diverse bioprocesses involved in uptake and conversion of xylose sugars from agricultural residues and industrial effluents. A comprehensive report on xylitol occurrence and biotechnological production processes with varied substrates has been encompassed. KEY POINTS: • Xylitol from agro-industrial waste • Microbial xylose assimilation.

Molecular and structural characterization of a promiscuous chalcone synthase from the fern species Stenoloma chusanum.

The key enzymes involved in the flavonoid biosynthesis pathway have been extensively studied in seed plants, but relatively less in ferns. In this study, two 4-Coumarate: coenzyme A ligases (Sc4CL1 and Sc4CL2) and one novel chalcone synthase (ScCHS1) were functionally characterized by mining the Stenoloma chusanum transcriptome database. Recombinant Sc4CLs were able to esterify various hydroxycinnamic acids to corresponding acyl-coenzyme A (CoA). ScCHS1 could catalyze p-coumaroyl-CoA, cinnamoyl-CoA, caffeoyl-CoA, and feruloyl-CoA to form naringenin, pinocembrin, eriodictyol, and homoeriodictyol, respectively. Moreover, enzymatic kinetics studies revealed that the optimal substrates of ScCHS1 were feruloyl-CoA and caffeoyl-CoA, rather than p-coumaroyl-CoA, which was substantially different from the common CHSs. Crystallographic and site-directed mutagenesis experiments indicated that the amino acid residues, Leu87, Leu97, Met165, and Ile200, located in the substrate-binding pocket near the B-ring of products, could exert a significant impact on the unique catalytic activity of ScCHS1. Furthermore, overexpression of ScCHS1 in tt4 mutants could partially rescue the mutant phenotypes. Finally, ScCHS1 and Sc4CL1 were used to synthesize flavanones and flavones with multi-substituted hydroxyl and methoxyl B-ring in Escherichia coli, which can effectively eliminate the need for the cytochrome P450 hydroxylation/O-methyltransferase from simple phenylpropanoid acids. In summary, the identification of these important Stenoloma enzymes provides a springboard for the future production of various flavonoids in E. coli.

Application of quorum sensing system in microbial synthesis of valuable chemicals: a mini-review.

With advantages of low substrates cost, high optical purity of end products and environmentally friendly fermentation process, microbial production of valuable chemicals grow rapidly. Compared with static microbial strain engineering strategies, such as gene deletion, overexpression and mutation, dynamic pathway regulation is a new approach that balances cellular growth and chemical production. Quorum sensing is a natural microbial communication system responsible for cell-density-related cell behaviors. Accordingly, quorum sensing systems can be employed to achieve dynamic regulation in microorganisms without the need for manual intervention or the use of chemical inducers. In this review, natural quorum sensing systems are firstly summarized. Then, recent progress in using quorum sensing circuits in the field of metabolic engineering is highlighted. The current application challenges of quorum sensing systems and future perspectives in microbial synthesis of chemicals are also discussed.

Production of Coenzyme Q10 by microbes: an update.

Coenzyme Q10 (CoQ10) is the main CoQ species in human and is used extensively in food, cosmetic and medicine industries because of its antioxidant properties and its benefit in prophylactic medicine and therapy for a variety of diseases. Among various approaches to increase the production of CoQ10, microbial fermentation is the most effective. As knowledge of the biosynthetic enzymes and regulatory mechanisms modulating CoQ10 production increases, opportunities arise for metabolic engineering of CoQ10 in microbial hosts. In this review, we present various strategies used up to date to improve CoQ10 production and focus on metabolic engineering of CoQ10 overproduction in microbes. General strategies of metabolic engineering include providing sufficient precursors for CoQ10, increasing metabolic fluxes, and expanding storage capacity for CoQ10. Based on these strategies, CoQ10 production has been significantly improved in natural CoQ10 producers, as well as in heterologous hosts.

Effectiveness of recombinant Escherichia coli on the production of (R)-(+)-perillyl alcohol.

BACKGROUND: (R)-(+)-perillyl alcohol is a naturally oxygenated monoterpene widely used as the natural flavor additives, insecticides, jet fuels and anti-cancer therapies. It was also readily available monoterpene precursors. However, this natural product is present at low concentrations from plant sources which are not economically viable. Therefore, alternative microbial production methods are rapidly emerging as an attractive alternative to make (R)-(+)-perillyl alcohol production more sustainable and environmentally friendly. RESULTS: We engineered Escherichia coli to possess a heterologous mevalonate (MVA) pathway, including limonene synthase, P-cymene monoxygenase hydroxylase and P-cymene monoxygenase reductase for the production of (R)-(+)-perillyl alcohol. The concentration of (R)-(+)-limonene (the monoterpene precursor to (R)-(+)-perillyl alcohol) reached 45 mg/L from glucose. Enhanced (R)-(+)-perillyl alcohol production was therefore achieved. The strain produced (R)-(+)-perillyl alcohol at a titer of 87 mg/L and a yield of 1.5 mg/g glucose in a 5 L bioreactor fed batch system. CONCLUSIONS: These datas highlight the efficient production of (R)-(+)-perillyl alcohol through the mevalonate pathway from glucose. This method serves as a platform for the future production of other monoterpenes.

Microbial production of gamma-aminobutyric acid: applications, state-of-the-art achievements, and future perspectives.

Gamma-aminobutyric acid (GABA) is an important non-protein amino acid with wide-ranging applications. Currently, GABA can be produced by a variety of methods, including chemical synthesis, plant enrichment, enzymatic methods, and microbial production. Among these methods, microbial production has gained increasing attention to meet the strict requirements of an additive in the fields of food, pharmaceutical, and livestock. In addition, renewable and abundant resources, such as glucose and lignocellulosic biomass can also be used for GABA microbial production under mild and environmentally friendly processing conditions. In this review, the applications, metabolic pathways and physiological functions of GABA in different microorganisms were firstly discussed. A comprehensive overview of the current status of process engineering strategies for enhanced GABA production, including fermentation optimization and whole-cell conversion from different feedstocks by various host strains is also provided. We also presented the state-of-the-art achievements in strain development strategies for industrial lactic acid bacteria (LAB), Corynebacterium glutamicum and Escherichia coli to enhance the performance of GABA bioproduction. In order to use bio-based GABA in the fields of food and pharmaceutical, some Generally Recognized as Safe (GRAS) strains such as LAB and C. glutamicum will be the promising chassis hosts. Toward the end of this review, current challenges and valuable research directions/strategies on the improvements of process and strain engineering for economic microbial production of GABA are also suggested.

Study on the isoprene-producing co-culture system of Synechococcus elongates-Escherichia coli through omics analysis.

BACKGROUND: The majority of microbial fermentations are currently performed in the batch or fed-batch manner with the high process complexity and huge water consumption. The continuous microbial production can contribute to the green sustainable development of the fermentation industry. The co-culture systems of photo-autotrophic and heterotrophic species can play important roles in establishing the continuous fermentation mode for the bio-based chemicals production. RESULTS: In the present paper, the co-culture system of Synechococcus elongates-Escherichia coli was established and put into operation stably for isoprene production. Compared with the axenic culture, the fermentation period of time was extended from 100 to 400 h in the co-culture and the isoprene production was increased to eightfold. For in depth understanding this novel system, the differential omics profiles were analyzed. The responses of BL21(DE3) to S. elongatus PCC 7942 were triggered by the oxidative pressure through the Fenton reaction and all these changes were linked with one another at different spatial and temporal scales. The oxidative stress mitigation pathways might contribute to the long-lasting fermentation process. The performance of this co-culture system can be further improved according to the fundamental rules discovered by the omics analysis. CONCLUSIONS: The isoprene-producing co-culture system of S. elongates-E. coli was established and then analyzed by the omics methods. This study on the co-culture system of the model S. elongates-E. coli is of significance to reveal the common interactions between photo-autotrophic and heterotrophic species without natural symbiotic relation, which could provide the scientific basis for rational design of microbial community.

Selection of the optimal tyrosine hydroxylation enzyme for (S)-reticuline production in Escherichia coli.

We have constructed an Escherichia coli-based platform producing (S)-reticuline, an important intermediate of benzylisoquinoline alkaloids (BIAs), using up to 14 genes. (S)-reticuline was produced from a simple carbon source such as glucose and glycerol via L-DOPA, which is synthesized by hydroxylation of L-tyrosine, one of the rate-limiting steps of the reaction. There are three kinds of enzymes catalyzing tyrosine hydroxylation: tyrosinase (TYR), tyrosine hydroxylase (TH), and 4-hydroxyphenylacetate 3-monooxygenase (HpaBC). Here, to further improve (S)-reticuline production, we chose eight from these three kinds of tyrosine hydroxylation enzymes (two TYRs, four THs, and two HpaBCs) derived from various organisms, and examined which enzyme was optimal for (S)-reticuline production in E. coli. TH from Drosophila melanogaster was the most suitable for (S)-reticuline production under the experimental conditions tested. We improved the productivity by genome integration of a gene set for L-tyrosine overproduction, introducing the regeneration pathway of BH4, a cofactor of TH, and methionine addition to enhance the S-adenosylmethionine supply. As a result, the yield of (S)-reticuline reached up to 384 µM from glucose in laboratory-scale shake flask. Furthermore, we found three inconsistent phenomena: an inhibitory effect due to additional gene expression, conflicts among the experimental conditions, and interference of an upstream enzyme from an additional downstream enzyme. Based on these results, we discuss future perspectives and challenges of integrating multiple enzyme genes for material production using microbes. Graphical abstract The optimal tyrosine hydroxylation enzyme for (S)-reticuline production in Escherichia coli KEY POINTS: • There are three types of enzymes catalyzing tyrosine hydroxylation reaction: tyrosinase, tyrosine hydroxylase, and 4-hydroxyphenylacetate 3-monooxygenase. • Tyrosine hydroxylase from Drosophila melanogaster exhibited the highest activity and was suitable for (S)-reticuline production in E. coli. • New insights were provided on constructing an alkaloid production system with multi-step reactions in E. coli.