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D-Mannose 2-epimerase (MEase) catalyzes the bioconversion between D-glucose and D-mannose. It is an important potential biocatalyst for large-scale production of D-mannose, a functional monosaccharide used in pharmaceutical and food industries. In this study, a new microbial MEase was characterized from Runella zeae DSM 19591. The enzyme was purified by one-step nickel-affinity chromatography and determined to be a dimeric protein with two identical subunits of approximately 86.1 kDa by gel filtration. The enzyme showed the highest activity at pH 8.0 and 40 °C, with a specific activity of 2.99 U/mg on D-glucose and 3.71 U/mg on D-mannose. The melting temperature (Tm) was 49.4 °C and the half-life was 115.14 and 3.23 h at 35 and 40 °C, respectively. The purified enzyme (1 U/mL) produced 115.7 g/L of D-mannose from 500 g/L of D-glucose for 48 h, with a conversion ratio of 23.14 %. It was successfully expressed in Bacillus subtilis WB600 via pP43NMK as the vector. The highest fermentation activity was 10.58 U/mL after fed-batch cultivation for 28 h, and the whole cells of recombinant B. subtilis produced 114.0 g/L of D-mannose from 500 g/L of D-glucose, with a conversion ratio of 22.8 %.

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BACKGROUND: Recombinant peptide production in Escherichia coli provides a sustainable alternative to environmentally harmful and size-limited chemical synthesis. However, in-vivo production of disulfide-bonded peptides at high yields remains challenging, due to degradation by host proteases/peptidases and the necessity of translocation into the periplasmic space for disulfide bond formation. RESULTS: In this study, we established an expression system for efficient and soluble production of disulfide-bonded peptides in the periplasm of E. coli. We chose model peptides with varying complexity (size, structure, number of disulfide bonds), namely parathyroid hormone 1-84, somatostatin 1-28, plectasin, and bovine pancreatic trypsin inhibitor (aprotinin). All peptides were expressed without and with the N-terminal, low molecular weight CASPON™ tag (4.1 kDa), with the expression cassette being integrated into the host genome. During BioLector™ cultivations at microliter scale, we found that most of our model peptides can only be sufficiently expressed in combination with the CASPON™ tag, otherwise expression was only weak or undetectable on SDS-PAGE. Undesired degradation by host proteases/peptidases was evident even with the CASPON™ tag. Therefore, we investigated whether degradation happened before or after translocation by expressing the peptides in combination with either a co- or post-translational signal sequence. Our results suggest that degradation predominantly happened after the translocation, as degradation fragments appeared to be identical independent of the signal sequence, and expression was not enhanced with the co-translational signal sequence. Lastly, we expressed all CASPON™-tagged peptides in two industry-relevant host strains during C-limited fed-batch cultivations in bioreactors. We found that the process performance was highly dependent on the peptide-host-combination. The titers that were reached varied between 0.6-2.6 g L-1, and exceeded previously published data in E. coli. Moreover, all peptides were shown by mass spectrometry to be expressed to completion, including full formation of disulfide bonds. CONCLUSION: In this work, we demonstrated the potential of the CASPON™ technology as a highly efficient platform for the production of soluble peptides in the periplasm of E. coli. The titers we show here are unprecedented whenever parathyroid hormone, somatostatin, plectasin or bovine pancreatic trypsin inhibitor were produced in E. coli, thus making our proposed upstream platform favorable over previously published approaches and chemical synthesis.

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Amino oligosaccharides (AOs) possess various biological activities and are valuable in the pharmaceutical, food industries, and agriculture. However, the industrial manufacturing of AOs has not been realized yet, despite reports on physical, chemical, and biological approaches. In this study, the de novo production of chitin oligosaccharides (CHOS), a type of structurally defined AOs, was achieved in Escherichia coli through combinatorial pathway engineering. The most suitable glycosyltransferase for CHOS production was found to be NodCL from Mesorhizobium Loti. Then, by knocking out the nagB gene to block the flow of N-acetyl-d-glucosamine (NAG) to the glycolytic pathway in E. coli and adjusting the copy number of NodCL-coding gene, the CHOS yield was increased by 6.56 times. Subsequently, by introducing of UDP-N-acetylglucosamine (UDP-GlcNAc) salvage pathway for and optimizing fermentation conditions, the yield of CHOS reached 207.1 and 468.6 mg/L in shake-flask cultivation and a 5-L fed-batch bioreactor, respectively. Meanwhile, the concentration of UDP-GlcNAc was 91.0 mg/L, the highest level reported in E. coli so far. This study demonstrated, for the first time, the production of CHOS with distinct structures in plasmid-free E. coli, laying the groundwork for the biosynthesis of CHOS and providing a starting point for further engineering and commercial production.

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We report the successful fabrication of a pharmaceutical cellular bank (PCB) containing magnetotactic bacteria (MTB), which belong to the Magnetospirillum gryphiswaldense MSR1 species. To produce such PCB, we amplified MTB in a minimal growth medium essentially devoid of other heavy metals than iron and of CMR (Carcinogenic, mutagenic and reprotoxic) products. The PCB enabled to acclimate MTB to such minimal growth conditions and then to produce highly pure magnetosomes composed of more than 99.9% of iron. The qualification of the bank as a PCB relies first on a preserved identity of the MTB compared with the original strain, second on genetic bacterial stability observed over 100 generations or under cryo-preservation for 16 months, third on a high level of purity highlighted by an absence of contaminating microorganisms in the PCB. Furthermore, the PCB was prepared under high-cell load conditions (9.108 cells/mL), allowing large-scale bacterial amplification and magnetosome production. In the future, the PCB could therefore be considered for commercial as well as research orientated applications in nanomedicine. We describe for the first-time conditions for setting-up an effective pharmaceutical cellular bank preserving over time the ability of certain specific cells, i.e. Magnetospirillum gryphiswaldense MSR1 MTB, to produce nano-minerals, i.e. magnetosomes, within a pharmaceutical setting.

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The growing commercial application of microalgae in different industry sectors, including the production of bioenergy, pharmaceuticals, nutraceuticals, chemicals, feed, and food, demands large quantities of microalgal biomass with specific compositions produced at reasonable prices. Extensive studies have been carried out on the design of new and improvement of current cultivation systems and the optimisation of growth medium composition for high productivity of microalgal biomass. In this study, the concentrations of the main macronutrients, silicon, nitrogen and phosphorus, essential for the growth of diatom Nitzschia sp. S5 were optimised to obtain a high biomass concentration. The effect of main macronutrients on growth kinetics and cell composition was also studied. Silicon had the most significant effect on diatom growth during batch cultivation. The concentration of biomass increased 5.45-fold (0.49 g L-1) at 1 mM silicon concentration in modified growth medium compared to the original Guillard f/2 medium. Optimisation of silicon, nitrogen, and phosphorus quantities and ratios further increased biomass concentration. The molar ratio of Si:N:P = 7:23:1 mol:mol:mol yielded the highest biomass concentration of 0.73 g L-1. Finally, the fed-batch diatom cultivation of diatom using an optimised Guillard f/2 growth medium with four additions of concentrated macronutrient solution resulted in 1.63 g L-1 of microalgal biomass. The proteins were the most abundant macromolecules in microalgal biomass, with a lower content of carbohydrates and lipids under all studied conditions.

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2'-Fucosyllactose (2'-FL) which is well-known human milk oligosaccharide was biotechnologically synthesized using engineered Corynebacterium glutamicum, a GRAS microbial workhorse. By construction of the complete de novo pathway for GDP-L-fucose supply and heterologous expression of Escherichia coli lactose permease and Helicobacter pylori α-1,2-fucosyltransferase, bioengineered C. glutamicum BCGW_TL successfully biosynthesized 0.25 g L-1 2'-FL from glucose. The additional genetic perturbations including the expression of a putative 2'-FL exporter and disruption of the chromosomal pfkA gene allowed C. glutamicum BCGW_cTTLEΔP to produce 2.5 g L-1 2'-FL batchwise. Finally, optimized fed-batch cultivation of the BCGW_cTTLEΔP using glucose, fructose, and lactose resulted in 21.5 g L-1 2'-FL production with a productivity of 0.12 g L-1 •h, which were more than 3.3 times higher value relative to the batch culture of the BCGW_TL. Conclusively, it would be a groundwork to adopt C. glutamicum for biotechnological production of other food additives including human milk oligosaccharides.

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The continual rise in the consumption of petroleum-based synthetic polymers raised a significant environmental concern. Bacillus pseudomycoides SAS-B1 is a gram-positive rod-shaped halophilic bacterium capable of accumulating Polyhydroxybutyrate (PHB)-an intracellular biodegradable polymer. In the present study, the optimal conditions for cell cultivation in the seed media were developed. The optimal factors included a preservation age of 14 to 21 days (with 105 to 106 cells/mL), inoculum size of 0.1 % (w/v), 1 % (w/v) glucose, and growth temperature of 30 °C. The cells were then cultivated in a two-stage fermentation process utilizing glycerol and Corn Steep Liquor (CSL) as carbon and nitrogen sources, respectively. PHB yield was effectively increased from 2.01 to 9.21 g/L through intermittent feeding of glycerol and CSL, along with acrylic acid. FTIR, TGA, DSC, and XRD characterization studies were employed to enumerate the recovered PHB and determine its physicochemical properties. Additionally, the study assessed the cradle-to-gate Life Cycle Assessment (LCA) of PHB production, considering net CO2 generation and covering all major environmental impact categories. The production of 1000 kg of PHB resulted in lower stratospheric ozone depletion and comparatively reduced carbon dioxide emissions (2022.7 kg CO2 eq.) and terrestrial ecotoxicity (9.54 kg 1,4-DCB eq.) than typical petrochemical polymers.

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Intracellular hyperaccumulation of phycocyanin (PC) and its high susceptibility to degradation at higher temperatures are major challenging problems associated with its production from cyanobacteria. The present study evaluated different concentrations of organic acids (1, 2, and 3 mM) (citric acid, acetic acid, succinic acid, fumaric acid, and oxalic acid) under fed-batch mode on the biomass and phycobiliproteins' production from Arthrospira platensis. Besides they were evaluated at 2.5-7.5 mM as preservative to stabilize PC at high temperatures. The incorporation of 3 mM of succinic acid into the cultivation medium enhanced the biomass and PC productivity to 164.05 and 26.70 mg L-1 day-1, which was ~ 2- and threefold higher than control, respectively. The produced PC in this treatment was food-grade with a 2.2 purity ratio. The use of organic acids also enhanced the thermal stability of PC. Citric acid (7.5 mM) markedly promoted the half-life values of PC to 189.44 min compared to 71.84 min in the control. The thermodynamic analysis confirmed higher thermostability of PC in the presence of organic acids and indicated the endothermic and non-spontaneity of the thermal denaturation process. The findings of the present study confirmed that organic acids could be utilized as cost effective and sustainable compounds for promoting not only phycobiliproteins' production but also the thermostability of PC for potential application in food industry.

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Regulation of metabolic gene expression is crucial for maximizing bioproduction titers. Recent engineering tools including CRISPR/Cas9, CRISPR interference (CRISPRi), and CRISPR activation (CRISPRa) have enabled effective knock-out, knock-down, and overexpression of endogenous pathway genes, respectively, for advanced strain engineering. CRISPRi in particular has emerged as a powerful tool for gene repression through the use of a deactivated Cas9 (dCas9) protein and target guide RNA (gRNA). By constructing gRNA arrays, CRISPRi has the capacity for multiplexed gene downregulation across multiple orthogonal pathways for enhanced bioproduction titers. In this study, we harnessed CRISPRi to downregulate 32 essential and non-essential genes in E. coli strains heterologously expressing either the original mevalonate pathway or isopentenyl diphosphate (IPP) bypass pathway for isoprenol biosynthesis. Isoprenol remains a candidate bioproduct both as a drop-in blend additive and as a precursor for the high-performance sustainable aviation fuel, 1,4-dimethylcyclooctane (DMCO). Of the 32 gRNAs targeting genes associated with isoprenol biosynthesis, a subset was found to vastly improve product titers. Construction of a multiplexed gRNA library based on single guide RNA (sgRNA) performance enabled simultaneous gene repression, yielding a 3 to 4.5-fold increase in isoprenol titer (1.82 ± 0.19 g/L) on M9-MOPS minimal medium. We then scaled the best performing CRISPRi strain to 2-L fed-batch cultivation and demonstrated translatable titer improvements, ultimately obtaining 12.4 ± 1.3 g/L isoprenol. Our strategy further establishes CRISPRi as a powerful tool for tuning metabolic flux in production hosts and that titer improvements are readily scalable with potential for applications in industrial bioproduction.

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N-Acetylneuraminic acid (NeuAc) is the predominant sialic acid found in human cells and a human-identical milk monosaccharide. Due to its numerous health benefits, it has great commercial potential in the pharmaceutical, cosmetic, and food industries. Microbial synthesis via metabolic engineering strategies is an important approach to its large-scale production. In this study, a NeuAc synthetic pathway was constructed in Escherichia coli BL21(DE3) by deleting the competitive pathway genes and introducing two genes encoding UDP-N-acetylglucosamine (GlcNAc) 2-epimerase (NeuC) and NeuAc synthase (NeuB). UDP-GlcNAc pathway genes, glmS, glmM, and glmU, were overexpressed to strengthen precursor supply for enhancement of NeuAc synthesis. The microbial source of neuC and neuB was optimized, and their expression was fine-tuned. In addition, glycerol as the carbon source showed a much better effect on NeuAc synthesis than glucose. The final engineered strain produced 7.02 g/L NeuAc by shake-flask cultivation. The titer was enhanced to 46.92 g/L by fed-batch cultivation, with the productivity of 0.82 g/L/h and 1.05 g/g DCW.

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Ethyl acetate is an important organic solvent and currently produced from fossil carbon resources. Microbial synthesis of this ester from sugar-rich waste could be an interesting alternative. Therefore, synthesis of ethyl acetate by Kluyveromyces marxinanus DSM 5422 from delactosed whey permeate (DWP) was studied in an aerated stirred bioreactor at 40 °C. DWP is mainly composed of residual lactose and minerals. The minerals inhibited yeast growth, as witnessed by an increased lag period, a reduced growth rate, and an extended process duration. All experiments were therefore carried out with diluted DWP. In a series of batch experiments, the pH of iron-deficient DWP medium varied between 4.8 and 5.9. The pH of the cultivation medium significantly influenced cell growth and product syntheses, with the highest ethyl acetate yield of 0.347 g g-1 and lowest by-product formation achieved at pH 5.1. It is likely that this effect is due to pH-dependent iron chelation, which affects the iron bioavailability and the intracellular iron content, thus affecting growth and metabolite synthesis. The viability of yeast cells was always high despite the harsh conditions in DWP medium, which enabled extended usage of the biomass in repeated-batch and fed-batch cultivations. These two culture techniques increased the volume of DWP processed per time by 32 and 84% for the repeated-batch and the fed-batch cultivation, respectively, without a drop of the ester yield. KEY POINTS: • Delactosed whey permeate was converted to ethyl acetate with a high rate and yield. • The formation of ethyl acetate in DWP medium at iron limitation is pH-dependent. • Highly active yeasts from batch processes enabled extension as fed and repeated batch.

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Cinnamaldehyde (CAD) derived from cinnamon bark has received much attention for its potential as a nematicide and food additive. Previously, we have succeeded in developing an Escherichia coli strain (YHP05) capable of synthesizing cinnamaldehyde; however, the production titer (75 mg/L) was not sufficient for commercialization. Herein, to develop an economical and sustainable production bioprocess, we further engineered the YHP05 strain for non-auxotrophic, antibiotic-free, inducer-free hyperproduction of CAD using systematic metabolic engineering. First, the conversion of trans-cinnamic acid (t-CA) to CAD was improved by the co-expression of carboxylic acid reductase and phosphopantetheinyl transferase (PPTase) genes. Second, to prevent the spontaneous conversion of CAD to cinnamyl alcohol, 10 endogenous reductase and dehydrogenase genes were deleted. Third, all expression cassettes were integrated into the chromosomal DNA using an auto-inducible system for antibiotic- and inducer-free production. Subsequently, to facilitate CAD production, available pools of cofactors (NADPH, CoA, and ATP) were increased, and acetate pathways were deleted. With the final antibiotic-, plasmid-, and inducer-free strain (H-11MPmR), fed-batch cultivations combined with in situ product recovery (ISPR) were performed, and the production titer of CAD as high as 3.8 g/L could be achieved with 49.1 mg/L/h productivity, which is the highest CAD titer ever reported.

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l-Fucose is a natural deoxy hexose found in a variety of organisms. It possesses many physiological effects and has potential applications in pharmaceutical, cosmetic, and food industries. Microbial synthesis via metabolic engineering attracts increasing attention for efficient production of important chemicals. Previously, we reported the construction of a metabolically engineered Escherichia coli strain with high 2'-fucosyllactose productivity. Herein, we further introduced Bifidobacterium bifidum α-l-fucosidase via both plasmid expression and genomic integration and blocked the l-fucose assimilation pathway by deleting fucI, fucK, and rhaA. The highest l-fucose titers reached 6.31 and 51.05 g/L in shake-flask and fed-batch cultivation, respectively. l-Fucose synthesis was little affected by lactose added, and there was almost no 2'-fucosyllactose residue throughout the cultivation processes. The l-fucose productivity reached 0.76 g/L/h, indicating significant potential for large-scale industrial applications.

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Production of mushroom fruit bodies using farming technology could hardly meet the increasing demand of the world market. During the last several decades, there have been various basic and applied studies on fungal physiology, metabolism, process engineering, and (pre)clinical studies. The fundamental aspects of solid-state cultivation of various kinds of medicinal mushroom mycelia in various types of bioreactors were established. Solid-state cultivation of medicinal mushrooms for their biomass and bioactive metabolites production appear very suitable for veterinary use. Development of comprehensive submerged technologies using stirred tank and airlift bioreactors is the most promising technology for fast and large-scale production of medicinal fungi biomass and their pharmaceutically active products for human need. The potentials initiate the development of new drugs and some of the most attractive over-the-counter human and veterinary remedies. This article is to overview the engineering achievements in solid state and submerged cultivations of medicinal mushrooms in bioreactors.

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Despite the great potential for the industrial application of microalgae, production costs are still too high to make them a competitive raw material for commodities. Therefore, studying more efficient cultivation strategies in biomass production and economic viability is necessary. In this sense, this work aimed to reduce the production costs of biomass and biomolecules using phytohormone indole-3-acetic acid in different phases of Spirulina sp. LEB 18 cultivation. The experiments were conducted on bench scale indoor for 30 days. In each couple of experiments, the phytohormone was added on different days. The supplementation of indole-3-acetic acid on half of the growth deceleration phase of the microalga showed a cost reduction of 27%, 34%, and 75% for biomass, proteins, and carbohydrates, respectively. In addition, the strategy increased the final biomass concentration and carbohydrate content at 31.2 and 33.8%, respectively, compared to the condition without phytohormone. This study is the starting point for implementing phytohormone supplementation in industrial microalgal cultures.

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Fusion protein technologies to facilitate soluble expression, detection, or subsequent affinity purification in Escherichia coli are widely used but may also be associated with negative consequences. Although commonly employed solubility tags have a positive influence on titers, their large molecular mass inherently results in stochiometric losses of product yield. Furthermore, the introduction of affinity tags, especially the polyhistidine tag, has been associated with undesirable changes in expression levels. Fusion tags are also known to influence the functionality of the protein of interest due to conformational changes. Therefore, particularly for biopharmaceutical applications, the removal of the fusion tag is a requirement to ensure the safety and efficacy of the therapeutic protein. The design of suitable fusion tags enabling the efficient manufacturing of the recombinant protein remains a challenge. Here, we evaluated several N-terminal fusion tag combinations and their influence on product titer and cell growth to find an ideal design for a generic fusion tag. For enhancing soluble expression, a negatively charged peptide tag derived from the T7 bacteriophage was combined with affinity tags and a caspase-2 cleavage site applicable for CASPase-based fusiON (CASPON) platform technology. The effects of each combinatorial tag element were investigated in an integrated manner using human fibroblast growth factor 2 as a model protein in fed-batch lab-scale bioreactor cultivations. To confirm the generic applicability for manufacturing, seven additional pharmaceutically relevant proteins were produced using the best performing tag of this study, named CASPON-tag, and tag removal was demonstrated.

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2'-Fucosyllactose (2'-FL) is the most abundant oligosaccharide in human milk. In this study, a highly efficient biosynthetic route for 2'-FL production was designed via the de novo pathway of GDP-l-fucose using engineered Escherichia coli BL21(DE3). Specifically, plasmid-based strains with previously deleted lacZ and wcaJ were further reconstructed by introducing de novo pathway genes and α1,2-fucosyltransferase-encoding wbgL to realize 2'-FL synthesis. The 2'-FL titer was enhanced to 3.92 g/L by further introducing rcsA and rcsB. Subsequently, the additional wbgL expression cassette was chromosomally integrated into recA locus to strengthen fucosylation reaction and a strong constitutive promoter (PJ23119) was used to replace the original promoters of manC-manB and gmd-wcaG to improve 2'-FL synthesis. The maximal 2'-FL titer reached 9.06 and 79.23 g/L in shake-flask and fed-batch cultivation, respectively. The 2'-FL productivity reached 1.45 g/L/h, showing remarkable production potential in large-scale industrial application.

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3'-Sialyllactose (3'-SL) is one of the most important and simplest sialylated human milk oligosaccharides. In this study, a plasmid-based pathway optimization along with chromosomal integration strategies was applied for 3'-SL production. Specifically, the precursor CMP-Neu5Ac synthesis pathway genes and α2,3-sialyltransferase-encoding gene were introduced into Escherichia coli BL21(DE3)ΔlacZ to realize 3'-SL synthesis. Genes nanA and nanK involved in Neu5Ac catabolism were further deleted to reduce the metabolic flux of competitive pathway. Several α2,3-sialyltransferases from different species were selected to evaluate the sialylation effect. The precursor pools were balanced and improved by optimizing key enzyme expression involved in the UDP-GlcNAc and CMP-Neu5Ac synthesis pathway. Finally, an additional α2,3-sialyltransferase expression cassette was integrated into chromosome to maximize 3'-SL synthesis, and 4.5 g/L extracellular 3'-SL was produced at a shake-flask level. The extracellular 3'-SL concentration was raised to 23.1 g/L in a 5 L bioreactor fermentation, which represents the highest extracellular value ever reported.

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To date, most bio-based products of industrial biotechnology stem from sugar-based carbon sources originating from food and feed competing resources. Exemplary for bioproducts converted from glucose, the potential C5 platform chemical itaconic acid is presently produced by the filamentous fungus Aspergillus terreus. Here, an engineered strain of the industrial platform organism Corynebacterium glutamicum ATCC 13032 was used for acetate-based production of itaconic acid to overcome current production difficulties. For this purpose, C. glutamicum ICDR453C (pEKEx2-malEcadopt) with a mutated icd variant for reduced isocitrate dehydrogenase activity was constructed harbouring pEKEx2-malEcadopt, that includes a cis-aconitate dehydrogenase gene originating from A. terreus. Overall, a peak volumetric productivity of 1.01 gL-1h-1 was achieved resulting in an itaconate titer of 29.2 g/L, by using an integrated pH-coupled acetate feeding control in a fed-batch process without base titration. The results support the high potential of acetate as alternative substrate for bioproduction.

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l-Pipecolic acid is an important rigid cyclic nonprotein amino acid, which is obtained through the conversion of l-lysine catalyzed by l-lysine cyclodeaminase (LCD). To directly produce l-pipecolic acid from glucose by microbial fermentation, in this study, a recombinant Escherichia coli strain with high efficiency of l-pipecolic acid production was constructed. This study involves the dynamic regulation of the substrate concentration and the expression level of the l-lysine cyclodeaminase-coding gene pipA. In terms of substrate concentration, we adopted the l-lysine riboswitch to dynamically regulate the expression of lysP and lysO genes. As a result, the l-pipecolic acid yield was increased about 1.8-fold as compared with the control. In addition, we used chemically inducible chromosomal evolution (CIChE) to realize the presence of multiple copies of the pipA gene on the genome. The resultant E. coli strain XQ-11-4 produced 61 ± 3.4 g/L l-pipecolic acid with a productivity of 1.02 ± 0.06 g/(L·h) and a glucose conversion efficiency (α) of 29.6% in fermentation. This is the first report that discovered multiple copies of pipA gene expression on the genome that improves the efficiency of l-pipecolic acid production in an l-lysine high-producing strain, and these results give us new insight for constructing the other valuable biochemicals derived from l-lysine.

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