Identification of Indicator Genes for Agar Accumulation in Gracilariopsis lemaneiformis (Rhodophyta).

Agar, as a seaweed polysaccharide mainly extracted from Gracilariopsis lemaneiformis, has been commercially applied in multiple fields. To investigate factors indicating the agar accumulation in G. lemaneiformis, the agar content, soluble polysaccharides content, and expression level of 11 genes involved in the agar biosynthesis were analysed under 4 treatments, namely salinity, temperature, and nitrogen and phosphorus concentrations. The salinity exerted the greatest impact on the agar content. Both high (40‱) and low (10‱, 20‱) salinity promoted agar accumulation in G. lemaneiformis by 4.06%, 2.59%, and 3.00%, respectively. The content of agar as a colloidal polysaccharide was more stable than the soluble polysaccharide content under the treatments. No significant correlation was noted between the two polysaccharides, and between the change in the agar content and the relative growth rate of the algae. The expression of all 11 genes was affected by the 4 treatments. Furthermore, in the cultivar 981 with high agar content (21.30 ± 0.95%) compared to that (16.23 ± 1.59%) of the wild diploid, the transcriptional level of 9 genes related to agar biosynthesis was upregulated. Comprehensive analysis of the correlation between agar accumulation and transcriptional level of genes related to agar biosynthesis in different cultivation conditions and different species of G. lemaneiformis, the change in the relative expression level of glucose-6-phosphate isomerase II (gpiII), mannose-6-phosphate isomerase (mpi), mannose-1-phosphate guanylyltransferase (mpg), and galactosyltransferase II (gatII) genes was highly correlated with the relative agar accumulation. This study lays a basis for selecting high-yield agar strains, as well as for targeted breeding, by using gene editing tools in the future.

Understanding exopolysaccharide byproduct formation in Komagataella phaffii fermentation processes for recombinant protein production.

BACKGROUND: Komagataella phaffii (Pichia pastoris) has emerged as a common and robust biotechnological platform organism, to produce recombinant proteins and other bioproducts of commercial interest. Key advantage of K. phaffii is the secretion of recombinant proteins, coupled with a low host protein secretion. This facilitates downstream processing, resulting in high purity of the target protein. However, a significant but often overlooked aspect is the presence of an unknown polysaccharide impurity in the supernatant. Surprisingly, this impurity has received limited attention in the literature, and its presence and quantification are rarely addressed. RESULTS: This study aims to quantify this exopolysaccharide in high cell density recombinant protein production processes and identify its origin. In stirred tank fed-batch fermentations with a maximal cell dry weight of 155 g/L, the polysaccharide concentration in the supernatant can reach up to 8.7 g/L. This level is similar to the achievable target protein concentration. Importantly, the results demonstrate that exopolysaccharide production is independent of the substrate and the protein production process itself. Instead, it is directly correlated with biomass formation and proportional to cell dry weight. Cell lysis can confidently be ruled out as the source of this exopolysaccharide in the culture medium. Furthermore, the polysaccharide secretion can be linked to a mutation in the HOC1 gene, featured by all derivatives of strain NRRL Y-11430, leading to a characteristic thinner cell wall. CONCLUSIONS: This research sheds light on a previously disregarded aspect of K. phaffii fermentations, emphasizing the importance of monitoring and addressing the exopolysaccharide impurity in biotechnological applications, independent of the recombinant protein produced.

Biosynthetic production of anticoagulant heparin polysaccharides through metabolic and sulfotransferases engineering strategies.

Heparin is an important anticoagulant drug, and microbial heparin biosynthesis is a potential alternative to animal-derived heparin production. However, effectively using heparin synthesis enzymes faces challenges, especially with microbial recombinant expression of active heparan sulfate N-deacetylase/N-sulfotransferase. Here, we introduce the monosaccharide N-trifluoroacetylglucosamine into Escherichia coli K5 to facilitate sulfation modification. The Protein Repair One-Stop Service-Focused Rational Iterative Site-specific Mutagenesis (PROSS-FRISM) platform is used to enhance sulfotransferase efficiency, resulting in the engineered NST-M8 enzyme with significantly improved stability (11.32-fold) and activity (2.53-fold) compared to the wild-type N-sulfotransferase. This approach can be applied to engineering various sulfotransferases. The multienzyme cascade reaction enables the production of active heparin from bioengineered heparosan, demonstrating anti-FXa (246.09 IU/mg) and anti-FIIa (48.62 IU/mg) activities. This study offers insights into overcoming challenges in heparin synthesis and modification, paving the way for the future development of animal-free heparins using a cellular system-based semisynthetic strategy.

Study of two glycosyltransferases related to polysaccharide biosynthesis in Rhodococcus jostii RHA1.

The bacterial genus Rhodococcus comprises organisms performing oleaginous behaviors under certain growth conditions and ratios of carbon and nitrogen availability. Rhodococci are outstanding producers of biofuel precursors, where lipid and glycogen metabolisms are closely related. Thus, a better understanding of rhodococcal carbon partitioning requires identifying catalytic steps redirecting sugar moieties to storage molecules. Here, we analyzed two GT4 glycosyl-transferases from Rhodococcus jostii (RjoGlgAb and RjoGlgAc) annotated as α-glucan-α-1,4-glucosyl transferases, putatively involved in glycogen synthesis. Both enzymes were produced in Escherichia coli cells, purified to homogeneity, and kinetically characterized. RjoGlgAb and RjoGlgAc presented the "canonical" glycogen synthase activity and were actives as maltose-1P synthases, although to a different extent. Then, RjoGlgAc is a homologous enzyme to the mycobacterial GlgM, with similar kinetic behavior and glucosyl-donor preference. RjoGlgAc was two orders of magnitude more efficient to glucosylate glucose-1P than glycogen, also using glucosamine-1P as a catalytically efficient aglycon. Instead, RjoGlgAb exhibited both activities with similar kinetic efficiency and preference for short-branched α-1,4-glucans. Curiously, RjoGlgAb presented a super-oligomeric conformation (higher than 15 subunits), representing a novel enzyme with a unique structure-to-function relationship. Kinetic results presented herein constitute a hint to infer on polysaccharides biosynthesis in rhodococci from an enzymological point of view.

Chemical tools to track and perturb the expression of sialic acid and fucose monosaccharides.

The biosynthesis of glycans is a highly conserved biological process and found in all domains of life. The expression of cell surface glycans is increasingly recognized as a target for therapeutic intervention given the role of glycans in major pathologies such as cancer and microbial infection. Herein, we summarize our contributions to the development of unnatural monosaccharide derivatives to infiltrate and alter the expression of both mammalian and bacterial glycans and their therapeutic application.

Synthesis and mammalian cell compatibility of light-released glycan precursors for controlled metabolic engineering.

Sugar additions to biomolecules, or glycans, are some of the most abundant biomolecule modifications in biology because they enable cells to adapt to changing nutrient and stress conditions. An unmet challenge for the field of glycobiology is the study of glycan biosynthetic pathways with chemical control, especially in live cell settings. The objective of this study was to create biocompatible glycan precursors with controlled release properties. Here, we report eleven "caged" sugar probes that release glycan biosynthetic precursor molecules upon light exposure. The specific sugar pathways we target with our probes regulate the addition of the N-acetyl sugars GlcNAc, GalNAc, and sialic acid onto biomolecules in cells, each of which has the potential to alter glycan processes involved in cell morphology, signaling, and behavior. We hypothesized that our glycan precursor probes would remain biologically inert until light-initiated decaging conditions were met, avoiding biological activities including metabolism and cytotoxicity. The photocaged analogs of GlcNAc, GalNAc, and ManNAc (sialic acid precursor) sugars, which we call "photo-sugars," were released within minutes of light exposure at their optimal wavelengths. During the course of the study, we characterized the cell compatibility of these sugars under their respective decaging conditions, and found highly cell compatible GlcNAc, GalNAc, and ManNAc photocaged precursors. Release of GlcNAc-1-phosphate precursors led to altered ATP levels in cells, demonstrating preliminary metabolic engineering. We envision these probes as useful additions to the chemical glycobiology field that will enable spatiotemporal control over glycosylation pathways in living mammalian cells.

Chemical Synthesis and Evaluation of Exopolysaccharide Fragments Produced by Leuconostoc mesenteroides Strain NTM048.

Exopolysaccharides (EPSs) occur widely in natural products made by bacteria, fungi and algae. Some EPSs have intriguing biological properties such as anticancer and immunomodulatory activities. Our group has recently found that EPSs generated from Leuconostoc mesenteroides ssp. mesenteroides strain NTM048 (NTM048 EPS) enhanced a production of mucosal immunoglobulin A (IgA) of mouse. Herein, we described the synthesis and evaluation of the tetrasaccharide fragments of NTM048 EPS to obtain information about the molecular mechanism responsible for the IgA-inducing activity.

Role of genistein on the yield, structure and immunomodulatory activity of Monascus exopolysaccharides.

Manipulating the structures, physicochemical properties, and monosaccharide compositions of exopolysaccharides (EPS) isolated from microorganisms has been reported to enhance their biological activities. Hence, the aim of this work was to examine the effects of genistein addition during fermentation on the amount, physicochemical properties, and immunomodulatory activity of EPS secreted by M. purpureus. Results showed that genistein addition significantly increased M. purpureus biomass and EPS yield to 2.42 g L-1 and 6.08 g L-1, respectively, and affected the physicochemical properties and structures of EPS. Furthermore, EPS produced by genistein-treated M. purpureus (G-EMP) improved the immunomodulatory activity of RAW264.7 macrophages by increasing the secretion of nitric oxide and cytokines. Moreover, phospho-Jun N-terminal kinase (p-JNK), phospho-extracellular regulated protein kinase (p-ERK), phospho-p38 (p-p38) mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) phospho-p65 (p65) proteins were remarkably upregulated by G-EMP stimulation, blocking Toll-like receptor 4 (TLR4) that dramatically reduced the pinocytic and phagocytic capacities. Overall, these findings provide potential rationales for the application of genistein in improving the EPS yield of M. purpureus.

Cool Temperature Enhances Growth, Ferulic Acid and Flavonoid Biosynthesis While Inhibiting Polysaccharide Biosynthesis in Angelica sinensis.

Angelica sinensis, a perennial herb that produces ferulic acid and phthalides for the treatment of cardio-cerebrovascular diseases, prefers growing at an altitude of 1800-3000 m. Geographical models have predicted that high altitude, cool temperature and sunshade play determining roles in geo-authentic formation. Although the roles of altitude and light in yield and quality have been investigated, the role of temperature in regulating growth, metabolites biosynthesis and gene expression is still unclear. In this study, growth characteristics, metabolites contents and related genes expression were investigated by exposing A. sinensis to cooler (15 °C) and normal temperatures (22 °C). The results showed that plant biomass, the contents of ferulic acid and flavonoids and the expression levels of genes related to the biosynthesis of ferulic acid (PAL1, 4CLL4, 4CLL9, C3H, HCT, CCOAMT and CCR) and flavonoids (CHS and CHI) were enhanced at 15 °C compared to 22 °C. The contents of ligustilide and volatile oils exhibited slight increases, while polysaccharide contents decreased in response to cooler temperature. Based on gene expression levels, ferulic acid biosynthesis probably depends on the CCOAMT pathway and not the COMT pathway. It can be concluded that cool temperature enhances plant growth, ferulic acid and flavonoid accumulation but inhibits polysaccharide biosynthesis in A. sinensis. These findings authenticate that cool temperature plays a determining role in the formation of geo-authentic and also provide a strong foundation for regulating metabolites production of A. sinensis.

Statistical optimization of exopolysaccharide production by Leuconostoc pseudomesenteroides JF17 from native Atlantic Forest juçara fruit.

Leuconostoc pseudomesenteroides belongs to a group of lactic acid bacteria normally isolated from fruits, which has the capacity to produce exopolysaccharides (EPS). The present study aimed to optimize the EPS production of L. pseudomesenteroides JF17, isolated from juçara fruits (palm trees threatened with extinction in the Atlantic Forest), using the response surface methodology (RSM), besides evaluating the fermentation kinetics. The maximum production of EPS 53.77 mg/mL was obtained under ideal conditions of MRS broth supplemented with sucrose at 18%, w/v, fermentation temperature of 20 °C and initial pH of 7.30. The Luedeking-Piret model suggested that the production of EPS by the JF17 strain appeared to be associated with the cell growth of the microorganism, in addition to having high efficiency in the production of the polysaccharide from the substrate (Yp/s = 17.85 ± 0.74 mg EPS/log CFU ). Thus, the ideal optimization conditions and kinetic parameters can be useful for increasing the scale up of the fermentation process in the industrial production of EPS by L. pseudomesenteroides JF17.

Ecological and evolutionary diversification of sulphated polysaccharides in diverse photosynthetic lineages: A review.

Sulphated polysaccharides (SPs) are carbohydrate macromolecules with sulphate esters that are found among marine algae, seagrasses, mangroves and some terrestrial plants. The sulphate concentration in the ocean (28 mM) since ancient time could have driven the production of SPs in marine algae. SPs have a gelatinous property that can protect marine algae against desiccation and salinity stress. Agar and carrageenan are red algal SPs that are widely used as gelling agents in the food and pharmaceutical industries. The information on the SPs from freshwater and land plants are limited. In this review, we reviewed the taxonomic distribution and composition of SPs in different photosynthetic lineages, and explored the association of SP production in these diversified photosynthetic organisms with evolution history and environmental stresses. We also reviewed the genes/proteins involved in SP biosynthesis. Insights into SP biosynthetic machinery may shed light on the evolution that accompanied adaptation to life on earth.

Biosynthetic Mechanisms and Biological Significance of Glycerol Phosphate-Containing Glycan in Mammals.

Bacteria contain glycerol phosphate (GroP)-containing glycans, which are important constituents of cell-surface glycopolymers such as the teichoic acids of Gram-positive bacterial cell walls. These glycopolymers comprising GroP play crucial roles in bacterial physiology and virulence. Recently, the first identification of a GroP-containing glycan in mammals was reported as a variant form of O-mannosyl glycan on α-dystroglycan (α-DG). However, the biological significance of such GroP modification remains largely unknown. In this review, we provide an overview of this new discovery of GroP-containing glycan in mammals and then outline the recent progress in elucidating the biosynthetic mechanisms of GroP-containing glycans on α-DG. In addition, we discuss the potential biological role of GroP modification along with the challenges and prospects for further research. The progress in this newly identified glycan modification will provide insights into the phylogenetic implications of glycan.

Comparative Characteristics of the Expression of Fucosylated Glycans and Morphometric Parameters of Terminal Placental Villi Depending on the Severity of Preeclampsia.

We performed a comparative analysis of the expression of fucosylated glycans and morphometric characteristics of the terminal villi of the placenta, depending on the severity of preeclampsia (PE). Similar patterns of the expression of fucosylated glycans in the syncytiotrophoblast glycocalyx were revealed in the placental tissue of patients with normal pregnancy and with mild and severe PE: predominance of glycans with α1,6-fucose in the core, clustered fucose residues, and LeX glycan over α1,2-fucose-containing glycans. The expression pattern of fucosylated glycans and the composition of the endothelial glycocalyx are normally close to the expression pattern and composition of the syncytiotrophoblast glycocalyx; in case of mild and severe PE, the expression pattern of fucosylated glycans was changed uniformly, and α1,2-fucose-containing glycans significantly prevailed in the endothelial glycocalyx. According to the results of Fisher's LSD test, in patients with severe PE, the total vascular area in the villus prevailed over the indices established during physiological course of pregnancy (p=0.04) and mild PE (p=0.04). Correlation analysis revealed direct and reciprocal relationships between the morphometric characteristics of the terminal villi of the placenta and the expression of fucosylated glycans in the syncytiotrophoblast and endothelium in PE. Our results indicate a changed expression of fucosylated glycans in the glycocalyx of placental barrier structures and the morphometric parameters of villi in PE of different severity, which can affect the function of the placental barrier.

Complexation of europium(III) with exopolysaccharides from a marine bacterium envisaged as luminescent probe in a theranostic approach.

Exopolysaccharide (EPS) derivatives, produced by Alteromonas infernus bacterium, showed anti-metastatic properties in osteosarcoma (bone tumor). These EPSs could be employed as new drug delivery systems for therapeutic uses. They may represent a new class of ligands to be combined in a theranostic approach with fluorescent metals, such as Eu(III), to serve as imaging probe. The goal of this work was to investigate the feasibility of such coupling by time-resolved laser-induced fluorescence spectroscopy (TRLFS). Since these EPSs are polyelectrolytes their conformation could affect the complexation properties. Thus, viscosimetric measurements were performed as a function of their concentration as well as the background electrolyte concentration. Polysaccharides conformation exhibited a lower hydrodynamic volume for the highest ionic strengths. The resulting random-coiled conformation could affect the complexation with metal for high concentration but no change was evidenced when increasing europium concentration. Two sites of complexation of Eu(III) were evidenced by TRLFS in heparin, whereas only one site was evidenced in two modified EPSs produced from Alteromonas infernus.

Effect of surfactants on the production of polysaccharides from Schizophyllum commune through submerged fermentation.

Schizophyllum commune (S. commune) polysaccharides are biomacromolecules with multiple biological activities and wide applications. In this study, polysaccharide production through submerged fermentation of S. commune using different surfactants was investigated. The addition of 1 g/L of polyoxyethylene sorbitan monooleate (Tween 80) at the beginning of the fermentation showed the best promotional effects on collective exopolysaccharide (EPS) production (which increased by 37.17%) while shortening the production cycle by 2 days. The monosaccharide composition of the EPS produced when the added Tween 80 was similar to that of the control; however, the molecular weight (Mw) was lower. Notably, the addition of Tween 80 significantly increased the ATP levels and the transcription levels of phosphoglucomutase and ß-glucan synthase genes in the polysaccharide synthesis pathway. The addition of Tween 80 reduced the pellet size of the mycelium compared to that of the control, but did not significantly change the microstructure of the mycelial cells. This study proposes an efficient strategy for the production of polysaccharides through submerged fermentation of S. commune, and elucidates the detailed mechanism of using Tween 80 as a fermentation stimulatory reagent.

Production of Polysaccharides and Single-Cell Protein by Some Local Isolates of Trichoderma spp.

<b>Background and Objective:</b> Polysaccharides and Single-cell protein are one of the best essential natural products of microorganisms, they are excreted by different microorganisms such as yeast, fungi, bacteria and algae. This study was carried out to detect the ability of four local fungal isolates of <i>Trichoderma </i>spp. to produce polysaccharides and Single-cell protein. <b>Materials and Methods:</b> Standard Czapek Dox Broth Medium was used to detect the ability of fungal isolates to produce polysaccharides and Single-cell protein, with modified the components of medium for improved production using banana peels as a source of carbon and different nitrogen sources at different concentrations and the factorial experiment was carried out using a completely randomized design <b>Results:</b> The highest dry weight and polysaccharides production and protein content have been achieved for the fungus <i>T. reesei</i> with rates of (2.15, 0.276 and 0.94) g/100 mL, respectively, in comparison with the other treatments, the use of ammonium phosphate at concentration 0.6 g L¹ has given the highest dry weight and production of polysaccharides and protein content with rates of (3.75, 0.364 and 2.77) g/100 mL, respectively, also the use of banana peels extract at concentration 40 mL L¹ has given the highest dry weight and production of polysaccharides and protein content with rates of (5.21, 0.539 and 3.63) g/100 mL, respectively. <b>Conclusion:</b> The possibility of using the local isolate of <i>T. reesei</i> in the production of polysaccharides and Single-cell protein using some cheap agricultural waste such as banana peels as a carbon source instead of throwing them as waste and pollutants for the environment.

Functional Characterization of Two PLP-Dependent Enzymes Involved in Capsular Polysaccharide Biosynthesis from Campylobacter jejuni.

Campylobacter jejuni is a Gram-negative, pathogenic bacterium that causes campylobacteriosis, a form of gastroenteritis. C. jejuni is the most frequent cause of food-borne illness in the world, surpassing Salmonella and E. coli. Coating the surface of C. jejuni is a layer of sugar molecules known as the capsular polysaccharide that, in C. jejuni NCTC 11168, is composed of a repeating unit of d-glycero-l-gluco-heptose, d-glucuronic acid, d-N-acetyl-galactosamine, and d-ribose. The d-glucuronic acid moiety is further amidated with either serinol or ethanolamine. It is unknown how these modifications are synthesized and attached to the polysaccharide. Here, we report the catalytic activities of two previously uncharacterized, pyridoxal phosphate (PLP)-dependent enzymes, Cj1436 and Cj1437, from C. jejuni NCTC 11168. Using a combination of mass spectrometry and nuclear magnetic resonance, we determined that Cj1436 catalyzes the decarboxylation of l-serine phosphate to ethanolamine phosphate. Cj1437 was shown to catalyze the transamination of dihydroxyacetone phosphate to (S)-serinol phosphate in the presence of l-glutamate. The probable routes to the ultimate formation of the glucuronamide substructures in the capsular polysaccharides of C. jejuni are discussed.

Exopolysaccharides from microalgae: production, characterization, optimization and techno-economic assessment.

Microalgae cultivation for exopolysaccharide production has getting more attention as a result of their high hydrocarbon biosynthesis skill. The aim of this study is to examine the exopolysaccharide production potential of different species of microalgae. In this context, exopolysaccharides were produced from Chlorella minutissima, Chlorella sorokiniana and Botryococcus braunii microalgae and the effects of carbon and nitrogen content in the growth medium and illumination time on exopolysaccharide production were analyzed statistically using Box-Behnken experimental design. In addition, techno-economic assessment of exopolysaccharide production were also performed by using the most productive microalgae and optimum conditions determined in this study. As a result of the experiments, it was seen that C. minutissima, C. sorokiniana and B. braunii produced 0.245 ± 0.0025 g/L, 0.163 ± 0.0016 g/L and 0.117 ± 0.0007 g/L exopolysaccharide, respectively. Statistically, it was observed that there was an inverse relationship between the exopolysaccharide production and investigated parameters such as illumination period and carbon and nitrogen amounts of culture mediums. The techno-economic assessment comprising microalgal exopolysaccharide (EPS) bioprocess was carried out, and it showed that the system can be considered economically viable, yet can be improved with biorefinery approach.

Chronic in vitro fermentation and in vivo metabolism: Extracellular polysaccharides from Sporidiobolus pararoseus regulate the intestinal microbiome of humans and mice.

The fungus Sporidiobolus pararoseus not only produces carotenoids, but also produces bioactive extracellular polysaccharides (SPP). However, the relationship between SPP and the metabolism of gut microbiome is unclear. The aim of this study was to investigate the mechanism of SPP regulating intestinal health in vivo and in vitro. Results showed that SPP are nondigestible polysaccharides after the digestion with simulated stomach and small intestinal juice in vitro. After SPP was cultured in an in vitro intestinal simulation system for seven days, the concentration of short-chain fatty acids (SCFAs) increased; the microbial diversity changed; the relative abundance of Bifidobacterium and Streptococcus increased; and that of Escherichia Shigella and Lachnospiraceae NK4A136 decreased. In addition, metabolism of SPP by the mice colonic microbiome showed SPP decreased the relative abundance of Firmicutes and Bacteroidota, while the relative abundance of Verrucomicrobiota, Desulfobacterota, and Actinobacteriota increased. Finally, predicted Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolism results also showed that SPP can enhance the metabolism of cofactors, vitamins, amino acids, starch, and sucrose. In conclusion, SPP can multiply the intestinal beneficial bacteria of humans and mice, promote the production of SCFAs and metabolism of amino acids, and promote intestinal health.

Increased Production of Colanic Acid by an Engineered Escherichia coli Strain, Mediated by Genetic and Environmental Perturbations.

Colanic acid (CA) is a major exopolysaccharide synthesized by Escherichia coli that serves as a constituent of biofilm matrices. CA demonstrates potential applications in the food, cosmetics, and pharmaceutical industry. Moreover, L-fucose, a monomeric constituent of CA, exhibits various physiological activities, such as antitumor, anti-inflammatory, and skin-whitening. Here, the effects of genetic and environmental perturbations were investigated for improving CA production by E. coli. When rcsF, a positive regulator gene of CA synthesis, was expressed in E. coli ΔwaaF, a CA-producing strain constructed previously, the CA titer increased to 3051.2 mg/L as compared to 2052.8 mg/L observed with E. coli ΔwaaF. Among the environmental factors tested, namely, osmotic and oxidative stresses and pH, pH was a primary factor that significantly improved CA production. When the pH of the culture medium of E. coli ΔwaaF + rcsF was maintained at 7, the CA titer significantly increased to 4351.6 mg/L. The CA yield obtained with E. coli ΔwaaF + rcsF grown at pH 7 was 5180.4 mg CA/g dry cell weight, which is the highest yield of CA reported so far. This engineered E. coli system with optimization of environmental conditions can be employed for fast and economically-feasible production of CA.