Breakthrough infection elicits hypermutated IGHV3-53/3-66 public antibodies with broad and potent neutralizing activity against SARS-CoV-2 variants including the emerging EG.5 lineages.

The rapid emergence of SARS-CoV-2 variants of concern (VOCs) calls for efforts to study broadly neutralizing antibodies elicited by infection or vaccination so as to inform the development of vaccines and antibody therapeutics with broad protection. Here, we identified two convalescents of breakthrough infection with relatively high neutralizing titers against all tested viruses. Among 50 spike-specific monoclonal antibodies (mAbs) cloned from their B cells, the top 6 neutralizing mAbs (KXD01-06) belong to previously defined IGHV3-53/3-66 public antibodies. Although most antibodies in this class are dramatically escaped by VOCs, KXD01-06 all exhibit broad neutralizing capacity, particularly KXD01-03, which neutralize SARS-CoV-2 from prototype to the emerging EG.5.1 and FL.1.5.1. Deep mutational scanning reveals that KXD01-06 can be escaped by current and prospective variants with mutations on D420, Y421, L455, F456, N460, A475 and N487. Genetic and functional analysis further indicates that the extent of somatic hypermutation is critical for the breadth of KXD01-06 and other IGHV3-53/3-66 public antibodies. Overall, the prevalence of broadly neutralizing IGHV3-53/3-66 public antibodies in these two convalescents provides rationale for novel vaccines based on this class of antibodies. Meanwhile, KXD01-06 can be developed as candidates of therapeutics against SARS-CoV-2 through further affinity maturation.

Metabolic engineering of Escherichia coli BL21 strain using simplified CRISPR-Cas9 and asymmetric homology arms recombineering.

BACKGROUND: The recent CRISPR-Cas coupled with λ recombinase mediated genome recombineering has become a common laboratory practice to modify bacterial genomes. It requires supplying a template DNA with homology arms for precise genome editing. However, generation of homology arms is a time-consuming, costly and inefficient process that is often overlooked. RESULTS: In this study, we first optimized a CRISPR-Cas genome engineering protocol in the Escherichia coli (E. coli) BL21 strain and successfully deleted 10 kb of DNA from the genome in one round of editing. To further simplify the protocol, asymmetric homology arms were produced by PCR in a single step with two primers and then purified using a desalting column. Unlike conventional homology arms that are prepared through overlapping PCR, cloning into a plasmid or annealing synthetic DNA fragments, our method significantly both shortened the time taken and reduced the cost of homology arm preparation. To test the robustness of the optimized workflow, we successfully deleted 26 / 27 genes across the BL21 genome. Noteworthy, gRNA design is important for the CRISPR-Cas system and a general heuristic gRNA design has been proposed in this study. To apply our established protocol, we targeted 16 genes and iteratively deleted 7 genes from BL21 genome. The resulting strain increased lycopene yield by ~ threefold. CONCLUSIONS: Our work has optimized the homology arms design for gene deletion in BL21. The protocol efficiently edited BL21 to improve lycopene production. The same workflow is applicable to any E. coli strain in which genome engineering would be useful to further increase metabolite production.

Mediating oxidative stress enhances α-ionone biosynthesis and strain robustness during process scaling up.

BACKGROUND: α-Ionone is highly valued in cosmetics and perfumery with a global usage of 100-1000 tons per year. Metabolic engineering by microbial fermentation offers a promising way to produce natural (R)-α-ionone in a cost-effective manner. Apart from optimizing the metabolic pathways, the approach is also highly dependent on generating a robust strain which retains productivity during the scale-up process. To our knowledge, no study has investigated strain robustness while increasing α-ionone yield. RESULTS: Built on our previous work, here, we further increased α-ionone yield to 11.4 mg/L/OD in 1 mL tubes by overexpressing the bottleneck dioxygenase CCD1 and re-engineering the pathway, which is > 65% enhancement as compared to our previously best strain. However, the yield decreased greatly to 2.4 mg/L/OD when tested in 10 mL flasks. Further investigation uncovered an unexpected inhibition that excessive overexpression of CCD1 was accompanied with increased hydrogen peroxide (H2O2) production. Excessive H2O2 broke down lycopene, the precursor to α-ionone, leading to the decrease in α-ionone production in flasks. This proved that expressing too much CCD1 can lead to reduced production of α-ionone, despite CCD1 being the rate-limiting enzyme. Overexpressing the alkyl hydroperoxide reductase (ahpC/F) partially solved this issue and improved α-ionone yield to 5.0 mg/L/OD in flasks by reducing oxidative stress from H2O2. The strain exhibited improved robustness and produced ~ 700 mg/L in 5L bioreactors, the highest titer reported in the literature. CONCLUSION: Our study provides an insight on the importance of mediating the oxidative stress to improve strain robustness and microbial production of α-ionone during scaling up. This new strategy may be inspiring to the biosynthesis of other high-value apocarotenoids such as retinol and crocin, in which oxygenases are also involved.

Micro-Image Strain Sensing Method for Displacement and Strain Measurement in One United Sensor.

Micro-image strain sensing (MISS) is an innovative technology to measure strain within a measurement range of ±8300 microstrains. It has been proved to be effective and satisfy all requirements in the field of structural health monitoring. However, there is still room for improvement and extend the range of measurement. In this paper, an improved method is proposed to increase the measurement range of strain and displacement. Moreover, various tests were conducted to verify the efficiency of the improved method. The results showed that the modified method is efficient and accurate and can be readily used to extend the measurement range of both strain and displacement. This research will likely help stimulate the development of multifunctional sensors to obtain abundant useful information about structures in the field of structural health monitoring. It will allow measuring strain and displacement, which require different levels of accuracy, using one unified sensor.

Microbial astaxanthin biosynthesis: recent achievements, challenges, and commercialization outlook.

Astaxanthin is a natural pigment, known for its strong antioxidant activity and numerous health benefits to human and animals. Its antioxidant activity is known to be substantially greater than ß-carotene and about a thousand times more effective than vitamin E. The potential health benefits have generated a growing commercial interest, and the escalating demand has prompted the exploration of alternative supply chain. Astaxanthin naturally occurs in many sea creatures such as trout, shrimp, and microalgae, some fungi, bacteria, and flowering plants, acting to protect hosts against environmental stress and adverse conditions. Due to the rapid growth and simple growth medium requirement, microbes, such as the microalga, Haematococcus pluvialis, and the fungus Xanthophyllomyces dendrorhous, have been developed to produce astaxanthin. With advances in metabolic engineering, non-carotenogenic microbes, such as Escherichia coli and Saccharomyces cerevisiae, have been purposed to produce astaxanthin and significant progress has been achieved. Here, we review the recent achievements in microbial astaxanthin biosynthesis (with reference to metabolic engineering strategies) and extraction methods, current challenges (technical and regulatory), and commercialization outlook. Due to greenness, sustainability, and dramatic cost reduction, we envision microbial synthesis of astaxanthin offers an alternative means of production (e.g. chemical synthesis) in the near future.

Systematic engineering for high-yield production of viridiflorol and amorphadiene in auxotrophic Escherichia coli.

Isoprenoids, widely used as pharmaceuticals, flavors and nutraceuticals, represent one of the largest groups of natural products. Yet the low availability of top-quality (enantiopure) products and high cost limit the wide application of many valuable terpenoids. An example being viridiflorol, currently used in cosmetics and personal care products, may have other unexplored applications (e.g. as insect repellents; anti-inflammatory supplements). Here, we systematically optimized an auxotrophic Escherichia coli to produce viridiflorol with transcription, translation, enzyme and strain engineering. The best strain achieved 25.7 g/L and a yield of 0.22 g-viridiflorol/g-glucose in 2.5 days. Statistical analysis revealed the correlation between viridiflorol yields with the transcriptional levels and translation initiation rates, which enabled better understanding of the isoprenoid pathway and guiding future strain optimization. As a proof-of-concept example, we applied the knowledge to amorphadiene, anti-malaria drug artemisinin precursor, achieved 30 g/L. Hence, this study paved the way for commercialization of microbial terpenoid production.

A "plug-n-play" modular metabolic system for the production of apocarotenoids.

Apocarotenoids, such as α-, ß-ionone, and retinol, have high commercial values in the food and cosmetic industries. The demand for natural ingredients has been increasing dramatically in recent years. However, attempts to overproduce ß-ionone in microorganisms have been limited by the complexity of the biosynthetic pathway. Here, an Escherichia coli-based modular system was developed to produce various apocarotenoids. Incorporation of enzyme engineering approaches (N-terminal truncation and protein fusion) into modular metabolic engineering strategy significantly improved α-ionone production from 0.5 mg/L to 30 mg/L in flasks, producing 480 mg/L of α-ionone in fed-batch fermentation. By modifying apocarotenoid genetic module, this platform strain was successfully re-engineered to produce 32 mg/L and 500 mg/L of ß-ionone in flask and bioreactor, respectively (>80-fold higher than previously reported). Similarly, 33 mg/L of retinoids was produced in flask by reconstructing apocarotenoid module, demonstrating the versatility of the "plug-n-play" modular system. Collectively, this study highlights the importance of the strategy of simultaneous modular pathway optimization and enzyme engineering to overproduce valuable chemicals in microbes.

Multienzyme Biosynthesis of Dihydroartemisinic Acid.

One-pot multienzyme biosynthesis is an attractive method for producing complex, chiral bioactive compounds. It is advantageous over step-by-step synthesis, as it simplifies the process, reduces costs and often leads to higher yield due to the synergistic effects of enzymatic reactions. In this study, dihydroartemisinic acid (DHAA) pathway enzymes were overexpressed in Saccharomyces cerevisiae, and whole-cell biotransformation of amorpha-4,11-diene (AD) to DHAA was demonstrated. The first oxidation step by cytochrome P450 (CYP71AV1) is the main rate-limiting step, and a series of N-terminal truncation and transcriptional tuning improved the enzymatic activity. With the co-expression of artemisinic aldehyde dehydrogenase (ALDH1), which recycles NADPH, a significant 8-fold enhancement of DHAA production was observed. Subsequently, abiotic conditions were optimized to further enhance the productivity of the whole-cell biocatalysts. Collectively, approximately 230 mg/L DHAA was produced by the multi-step whole-cell reaction, a ~50% conversion from AD. This study illustrates the feasibility of producing bioactive compounds by in vitro one-pot multienzyme reactions.

Efflux transporter engineering markedly improves amorphadiene production in Escherichia coli.

Metabolic engineering aims at altering cellular metabolism to produce valuable products at high yields and titers. Achieving high titers and productivity can be challenging if final products are largely accumulated intracellularly. A potential solution to this problem is to facilitate the export of these substances from cells by membrane transporters. Amorphadiene, the precursor of antimalarial drug artemisinin, is known to be secreted from Escherichia coli overexpressing the biosynthetic pathway. In order to assess the involvement of various endogenous efflux pumps in amorphadiene transport, the effects of single gene deletion of 16 known multidrug-resistant membrane efflux transporters were examined. The outer membrane protein TolC was found to be intimately involved in amorphadiene efflux. The overexpression of tolC together with ABC family transporters (macAB) or MFS family transporters (emrAB or emrKY) enhanced amorphadiene titer by more than threefold. In addition, the overexpression of transporters in the lipopolysaccharide transport system (msbA, lptD, lptCABFG) was found to improve amorphadiene production. As efflux transporters often have a wide range of substrate specificity, the multiple families of transporters were co-expressed and synergistic benefits were observed in amorphadiene production. This strategy of screening and then rationally engineering transporters can be used to improve the production of other valuable compounds in E. coli. Biotechnol. Bioeng. 2016;113: 1755-1763. © 2016 Wiley Periodicals, Inc.

Experimental design-aided systematic pathway optimization of glucose uptake and deoxyxylulose phosphate pathway for improved amorphadiene production.

Artemisinin is a potent antimalarial drug; however, it suffers from unstable and insufficient supply from plant source. Here, we established a novel multivariate-modular approach based on experimental design for systematic pathway optimization that succeeded in improving the production of amorphadiene (AD), the precursor of artemisinin, in Escherichia coli. It was initially found that the AD production was limited by the imbalance of glyceraldehyde 3-phosphate (GAP) and pyruvate (PYR), the two precursors of the 1-deoxy-D-xylulose-5-phosphate (DXP) pathway. Furthermore, it was identified that GAP and PYR could be balanced by replacing the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) with the ATP-dependent galactose permease and glucose kinase system (GGS) and this resulted in fivefold increase in AD titer (11 to 60 mg/L). Subsequently, the experimental design-aided systematic pathway optimization (EDASPO) method was applied to systematically optimize the transcriptional expressions of eight critical genes in the glucose uptake and the DXP and AD synthesis pathways. These genes were classified into four modules and simultaneously controlled by T7 promoter or its variants. A regression model was generated using the four-module experimental data and predicted the optimal expression ratios among these modules, resulting in another threefold increase in AD titer (60 to 201 mg/L). This EDASPO method may be useful for the optimization of other pathways and products beyond the scope of this study.

IL-1ß promotes esophageal squamous cell carcinoma growth and metastasis through FOXO3A by activating the PI3K/AKT pathway.

Esophageal cancer is a common type of cancer that poses a significant threat to human health. While the pro-inflammatory cytokine IL-1ß has been known to contribute to the development of various types of tumors, its role in regulating esophageal cancer progression has not been extensively studied. Our studies found that the expression of IL-1ß and FOXO3A was increased in esophageal squamous cell carcinoma (ESCC). IL-1ß not only increased the proliferation, migration, and invasion of two ESCC cell lines but also promoted tumor growth and metastasis in nude mice. We also observed that IL-1ß and FOXO3A regulated the process of epithelial-mesenchymal transition (EMT) and autophagy. The PI3K/AKT pathway was found to be involved in the changes of FOXO3A with the expression level of IL-1ß. The AKT agonist (SC79) reversed the reduction of FOXO3A expression caused by the knockdown of IL-1ß, indicating that IL-1ß plays a role through the PI3K/AKT/FOXO3A pathway. Furthermore, the knockdown of FOXO3A inhibited ESCC development and attenuated the pro-cancer effect of overexpressed IL-1ß. Targeting IL-1ß and FOXO3A may be potentially valuable for the diagnosis and treatment of ESCC.

High-Yield Biosynthesis of trans-Nerolidol from Sugar and Glycerol.

Isoprenoids, or terpenoids, have wide applications in food, feed, pharmaceutical, and cosmetic industries. Nerolidol, an acyclic C15 isoprenoid, is widely used in cosmetics, food, and personal care products. Current supply of nerolidol is mainly from plant extraction that is inefficient, costly, and of inconsistent quality. Here, we screened various nerolidol synthases from bacteria, fungi, and plants and found that the strawberry nerolidol synthase was most active in Escherichia coli. Through systematic optimization of the biosynthetic pathways, carbon sources, inducer, and genome editing, we constructed a series of deletion strains (single mutants ΔldhA, ΔpoxB, ΔpflB, and ΔtnaA; double mutants ΔadhE-ΔldhA; and triple mutants and beyond ΔadhE-ΔldhA-ΔpflB and ΔadhE-ΔldhA-ΔackA-pta) that produced high yields of 100% trans-nerolidol. In flasks, the highest nerolidol titers were 1.8 and 3.3 g/L in glucose-only and glucose-lactose-glycerol media, respectively. The highest yield reached 26.2% (g/g), >90% of the theoretic yield. In two-phase extractive fed-batch fermentation, our strain produced ∼16 g/L nerolidol within 4 days with about 9% carbon yield (g/g). In a single-phase fed-batch fermentation, the strain produced >6.8 g/L nerolidol in 3 days. To the best of our knowledge, our titers and productivity are the highest in the literature, paving the way for future commercialization and inspiring biosynthesis of other isoprenoids.

Cytochrome P450 Surface Domains Prevent the ß-Carotene Monohydroxylase CYP97H1 of Euglena gracilis from Acting as a Dihydroxylase.

Molecular biodiversity results from branched metabolic pathways driven by enzymatic regioselectivities. An additional complexity occurs in metabolites with an internal structural symmetry, offering identical extremities to the enzymes. For example, in the terpene family, ß-carotene presents two identical terminal closed-ring structures. Theses cycles can be hydroxylated by cytochrome P450s from the CYP97 family. Two sequential hydroxylations lead first to the formation of monohydroxylated ß-cryptoxanthin and subsequently to that of dihydroxylated zeaxanthin. Among the CYP97 dihydroxylases, CYP97H1 from Euglena gracilis has been described as the only monohydroxylase. This study aims to determine which enzymatic domains are involved in this regioselectivity, conferring unique monohydroxylase activity on a substrate offering two identical sites for hydroxylation. We explored the effect of truncations, substitutions and domain swapping with other CYP97 members and found that CYP97H1 harbours a unique N-terminal globular domain. This CYP97H1 N-terminal domain harbours a hydrophobic patch at the entrance of the substrate channel, which is involved in the monohydroxylase activity of CYP97H1. This domain, at the surface of the enzyme, highlights the role of distal and non-catalytic domains in regulating enzyme specificity.

ß-Cryptoxanthin Production in Escherichia coli by Optimization of the Cytochrome P450 CYP97H1 Activity.

Cytochromes P450, forming a superfamily of monooxygenases containing heme as a cofactor, show great versatility in substrate specificity. Metabolic engineering can take advantage of this feature to unlock novel metabolic pathways. However, the cytochromes P450 often show difficulty being expressed in a heterologous chassis. As a case study in the prokaryotic host Escherichia coli, the heterologous synthesis of ß-cryptoxanthin was addressed. This carotenoid intermediate is difficult to produce, as its synthesis requires a monoterminal hydroxylation of ß-carotene whereas most of the classic carotene hydroxylases are dihydroxylases. This study was focused on the optimization of the in vivo activity of CYP97H1, an original P450 ß-carotene monohydroxylase. Engineering the N-terminal part of CYP97H1, identifying the matching redox partners, defining the optimal cellular background and adjusting the culture and induction conditions improved the production by 400 times compared to that of the initial strain, representing 2.7 mg/L ß-cryptoxanthin and 20% of the total carotenoids produced.

Mutagenesis of Dimer Interfacial Residues Improves the Activity and Specificity of Methyltransferase for cis-α-Irone Biosynthesis.

Promiscuous enzymes show great potential to establish new-to-nature pathways and expand chemical diversity. Enzyme engineering strategies are often employed to tailor such enzymes to improve their activity or specificity. It is paramount to identify the target residues to be mutated. Here, by exploring the inactivation mechanism with the aid of mass spectrometry, we have identified and mutated critical residues at the dimer interface region of the promiscuous methyltransferase (pMT) that converts psi-ionone to irone. The optimized pMT12 mutant showed ∼1.6-4.8-fold higher kcat than the previously reported best mutant, pMT10, and increased the cis-α-irone percentage from ∼70 to ∼83%. By one-step biotransformation, ∼121.8 mg L-1 cis-α-irone was produced from psi-ionone by the pMT12 mutant. The study offers new opportunities to engineer enzymes with enhanced activity and specificity.

Early Diagnosis and Prediction of Death Risk in Patients with Sepsis by Combined Detection of Serum PCT, BNP, Lactic Acid, and Apache II Score.

In order to investigate the expression levels of procalcitonin (PCT), B-type brain natriuretic peptide (BNP), and lactic acid (Lac) in serum of patients with sepsis, a retrospective analysis is conducted. 80 sepsis patients admitted to the ICU of our hospital from January 2019 to June 2020 are selected, and the application value of these factors combined with Apache II score in early diagnosis and prediction of death risk is analyzed. All patients are classified into survival group (n = 57) and death group (n = 23), and examined by blood routine. Lac, PCT, and BNP, and the serum PCT, BNP, and Lac levels were compared between the nonsepsis group and the control group. Furthermore, Acute Physiology and Chronic Health Status scoring System II (Apache II) is applied to evaluate the score difference between the sepsis group and the control group. The ROC curve demonstrates that PCT, BNP, and Lac combined with Apache II score can obtain high value for early diagnosis of sepsis. Compared with nonsepsis patients, the scores of serum Lac, PCT, and BNP and Apache II are significantly higher in sepsis patients. It is clearly evident that the combined detection of those indicators is valuable for early diagnosis and prediction of death, and will be suitable for widespread clinical application.

SOFA Score in relation to Sepsis: Clinical Implications in Diagnosis, Treatment, and Prognostic Assessment.

Purpose: To analyze the clinical significance of the sequential organ failure assessment (SOFA) score in the diagnosis, treatment, and prognostic assessment of sepsis. Methods: 140 patients with sepsis from January 2020 to January 2021 were selected as the observation group, and 40 healthy people were selected as the control group. The observation group was divided into mild group, severe group, and septic shock group by single blind grouping according to the condition of the disease, and they were also divided into survival group and death group according to the prognosis. Collect the fasting venous blood of the subjects in each group in the morning, compare the levels of total bilirubin (TBIL), blood creatinine (CR), and platelet count (PLT) in each group, and record and compare the patients' respiratory system oxygen partial pressure/inhaled oxygen concentration (po2/fio2), acute physiology and chronic health scoring system II (APACHE II), sequential organ failure assessment (sofa) score, q-SOFA score, and △SOFA score; Pearson analysis was used to analyze the correlation between SOFA score and other indicators; multivariate logistic regression was used to analyze the prognostic risk factors of patients with sepsis; receiver-operating characteristic curve (ROC) was used to analyze the value of SOFA score alone and in combination in the diagnosis, condition, and prognosis of sepsis. Results: There were significant differences in Apache II score, SOFA score, q-SOFA score map, po2/fio2, PLT, GCS, TBIL, and serum creatinine (SCR) between the control group and the observation group (P < 0.05). There were significant differences in Apache II score, SOFA score, q-SOFA score, mean arterial pressure (map) po2/fio2, PLT, Glasgow Coma Score (GCS), TBIL, SCR, and △SOFA score among patients in mild, severe, and septic shock groups (P < 0.05). There were significant differences in age, Apache II score, SOFA score, q-SOFA score, map, po2/fio2, PLT, GCS, TBIL, SCR, and △SOFA score between survival group and death group (P < 0.05). SOFA score and q-SOFA score were significantly positively correlated with TBIL and SCR and significantly negatively correlated with po2/fio2 and PLT; △SOFA score was significantly negatively correlated with TBIL and SCR and significantly positively correlated with map, po2/fio2, PLT, and GCS. Apache II score, SOFA score, and q-SOFA score were independent risk factors for sepsis patients, and △SOFA score, po2/fio2, and GCS score were protective factors (P < 0.05). ROC curve analysis showed that the AUC of sepsis combined with SOFA score and q-SOFA score was 0.880; the AUC of sepsis assessed by SOFA score, q-SOFA score, and △SOFA score was 0.929; the AUC of sepsis prognosis assessed by SOFA score, q-SOFA score, and △SOFA score was 0.900. Conclusion: SOFA score, q-SOFA score, and △SOFA score were abnormally expressed in patients with sepsis and were risk factors for the severity of the patient's condition and prognosis. The SOFA score, q-SOFA score, and △SOFA score were risk factors for the severity and prognosis of patients with sepsis and had some value in diagnosing sepsis and assessing the condition and prognosis, of which the combined value of the three was higher.

Total enzymatic synthesis of cis-α-irone from a simple carbon source.

Metabolic engineering has become an attractive method for the efficient production of natural products. However, one important pre-requisite is to establish the biosynthetic pathways. Many commercially interesting molecules cannot be biosynthesized as their native biochemical pathways are not fully elucidated. Cis-α-irone, a top-end perfumery molecule, is an example. Retrobiosynthetic pathway design by employing promiscuous enzymes provides an alternative solution to this challenge. In this work, we design a synthetic pathway to produce cis-α-irone with a promiscuous methyltransferase (pMT). Using structure-guided enzyme engineering strategies, we improve pMT activity and specificity towards cis-α-irone by >10,000-fold and >1000-fold, respectively. By incorporating the optimized methyltransferase into our engineered microbial cells, ~86 mg l-1 cis-α-irone is produced from glucose in a 5 l bioreactor. Our work illustrates that integrated retrobiosynthetic pathway design and enzyme engineering can offer opportunities to expand the scope of natural molecules that can be biosynthesized.

Biotechnological applications of S-adenosyl-methionine-dependent methyltransferases for natural products biosynthesis and diversification.

In the biosynthesis of natural products, methylation is a common and essential transformation to alter molecules' bioavailability and bioactivity. The main methylation reaction is performed by S-adenosylmethionine (SAM)-dependent methyltransferases (MTs). With advancements in genomic and chemical profiling technologies, novel MTs have been discovered to accept complex substrates and synthesize industrially valuable natural products. However, to achieve a high yield of small molecules in microbial hosts, many methyltransferase activities have been reported to be insufficient. Moreover, inadequate co-factor supplies and feedback inhibition of the by-product, S-adenosylhomocysteine (SAH), further limit MTs' activities. Here, we review recent advances in SAM-dependent MTs to produce and diversify natural products. First, we surveyed recently identified novel methyltransferases in natural product biosynthesis. Second, we summarized enzyme engineering strategies to improve methyltransferase activity, with a particular focus on high-throughput assay design and application. Finally, we reviewed innovations in co-factor regeneration and diversification, both in vitro and in vivo. Noteworthily, many MTs are able to accept multiple structurally similar substrates. Such promiscuous methyltransferases are versatile and can be tailored to design de novo pathways to produce molecules whose biosynthetic pathway is unknown or non-existent in nature, thus broadening the scope of biosynthesized functional molecules.

High-level de novo biosynthesis of glycosylated zeaxanthin and astaxanthin in Escherichia coli.

Because of wide applications in food, feed, pharmaceutical and cosmetic industries, the carotenoid market is growing rapidly. Most carotenoids are hydrophobic, which limits their bioavailability. Glycosylation is a natural route that substantially increases the water solubility, as well as the bioavailability, photostability and biological activities of carotenoids. Here, we report metabolic engineering efforts (e.g., promoter and RBS engineering, optimization of carbon sources and supplementation of bottleneck genes) to produce glycosylated carotenoids in Escherichia coli. By fine-tuning the carotenoid-biosynthetic genes (crtX, crtZ and crtY), our strain produced up to 47.2 mg/L (~ 11,670 ppm) of zeaxanthin glucosides, ~ 78% of the total carotenoids produced. In another construct with mevalonate, astaxanthin pathway and crtX genes, the strain produced a mixture of carotenoid glucosides including astaxanthin and adonixanthin glucosides with a total yield of 8.1 mg/L (1774 ppm). Our work demonstrated a proof-of-concept study for the microbial biosynthesis of glycosylated carotenoids.