Engineering Xylose Metabolism and Coutilization with Glucose in for Efficient Microbial Cell Factories.

Xylose, an important sugar component of lignocellulosic biomass, is often inefficiently utilized due to limitations in its transport and metabolic pathways. This study aimed to enhance the xylose metabolism in through metabolic engineering and transcriptional regulation strategies. First, the xylose assimilation pathway was constructed by the heterologous expression of xylose isomerase (XylA) and xylulose kinase (XylB), which improved the strain's xylose metabolic capability.

Site-specific Glycosylation via Metal-Targeted Conjugation Enhances Biochemical Functions of Bovine Lactoferrin.

Bovine lactoferrin (BLF) has attracted increasing attention due to its multiple nutritional benefits. However, the precise modification of BLF remains a significant challenge. Here, a site-specific glycosylation strategy, based on a copper assisted sequence-specific binding tag and prediction of glycosylation sites, was designed to enable the precise and controlled glycosylation of BLF in vitro.

Ancestral sequence reconstruction of a robust β-1,4-xylanase and efficient expression in Bacillus subtilis.

Xylanases are a class of glycoside hydrolases commonly used in the food, papermaking, and textile industries. However, most xylanases are rapidly inactivated under harsh industrial conditions. Here, a unique and robust GH11 xylanase, AncXyn18, was designed using an ancestral sequence reconstruction strategy, sequence analysis, structure prediction, and experimental verification.

A customized self-assembled synergistic biocatalyst for plastic depolymerization.

The enzymatic degradation of plastic offers a green, sustainable strategy and scalable circular carbon route for solving polyester waste. Among the earlies discovered plastic-degrading enzymes are PET hydrolase (PETase) and MHET hydrolase (MHETase), which act synergistically. To promote the adsorption of enzymes on PET surfaces, increase their robustness, and enable directly depolymerization, we designed hydrophobin HFBI fused-PETase and MHETase.

Multidimensional combinatorial screening for high-level production of erythritol in Yarrowia lipolytica.

Yarrowia lipolytica was successfully engineered to synthesize erythritol from crude glycerol, a cheap by-product of biodiesel production, but the yield remained low. Here, a biosensor-guided adaptive evolution screening platform was constructed to obtain mutant strains which could efficiently utilize crude glycerol to produce erythritol. Erythrose reductase D46A (M1) was identified as a key mutant through whole-genome sequencing of the strain G12, which exhibited higher catalytic activity (1.

High-efficiency chromosomal integrative amplification strategy for overexpressing α-amylase in Bacillus licheniformis.

Bacillus licheniformis is a well-known platform strain for production of industrial enzymes. However, the development of genetically stable recombinant B. licheniformis for high-yield enzyme production is still laborious.

Exploitation of ammonia-inducible promoters for enzyme overexpression in Bacillus licheniformis.

Ammonium hydroxide is conventionally used as an alkaline reagent and cost-effective nitrogen source in enzyme manufacturing processes. However, few ammonia-inducible enzyme expression systems have been described thus far. In this study, genomic-wide transcriptional changes in Bacillus licheniformis CBBD302 cultivated in media supplemented with ammonia were analyzed, resulting in identification of 1443 differently expressed genes, of which 859 genes were upregulated and 584 downregulated.

A novel aminopeptidase with potential debittering properties in casein and soybean protein hydrolysates.

A new aminopeptidase (An-APa) was identified and biochemically characterized from CICIM F0215. It had maximal activity at 40 °C and pH 7.0 and exhibited a broad substrate specificity both on hydrophilic and hydrophobic amino acid residues at N-terminals.

Biochemical characterization of two new aspartic proteases.

Two new aspartic proteases, PepAb and PepAc (encoded by and ), were heterologously expressed and biochemically characterized from F0215. They possessed a typical structure of pepsin-type aspartic protease with the conserved active residues D (84, 115), Y (131, 168) and D (281, 326), while their identity in amino acid sequences was only 19.0%.

Synthesis of flavor esters by a novel lipase from in a soybean-solvent system.

To find a lipase for synthesis of flavor esters in food processing, a total of 35 putative lipases from F0215 were heterologously expressed and their esterification properties in crude preparations were examined. One of them, named An-lipase with the highest esterification rate (23.1%) was selected for further study.

Improved ethanol productivity from lignocellulosic hydrolysates by Escherichia coli with regulated glucose utilization.

Background: Lignocellulosic ethanol could offer a sustainable source to meet the increasing worldwide demand for fuel. However, efficient and simultaneous metabolism of all types of sugars in lignocellulosic hydrolysates by ethanol-producing strains is still a challenge.

Results: An engineered strain Escherichia coli B0013-2021HPA with regulated glucose utilization, which could use all monosaccharides in lignocellulosic hydrolysates except glucose for cell growth and glucose for ethanol production, was constructed.

A novel strategy for production of ethanol and recovery of xylose from simulated corncob hydrolysate.

Objectives: To develop a xylose-nonutilizing Escherichia coli strain for ethanol production and xylose recovery.

Results: Xylose-nonutilizing E. coli CICIM B0013-2012 was successfully constructed from E.

Limitation of thiamine pyrophosphate supply to growing Escherichia coli switches metabolism to efficient D-lactate formation.

Efficient production of D-lactate by engineered Escherichia coli entails balancing cell growth and product synthesis. To develop a metabolic switch to implement a desirable transition from cell growth to product fermentation, a thiamine auxotroph B0013-080A was constructed in a highly efficient D-lactate producer E. coli strain B0013-070.

Highly efficient L-lactate production using engineered Escherichia coli with dissimilar temperature optima for L-lactate formation and cell growth.

Unlabelled: L-Lactic acid, one of the most important chiral molecules and organic acids, is produced via pyruvate from carbohydrates in diverse microorganisms catalyzed by an NAD+-dependent L-lactate dehydrogenase. Naturally, Escherichia coli does not produce L-lactate in noticeable amounts, but can catabolize it via a dehydrogenation reaction mediated by an FMN-dependent L-lactate dehydrogenase. In aims to make the E.

[High-efficiency L-lactate production from glycerol by metabolically engineered Escherichia coli].

High-efficient conversion of glycerol to L-lactate is beneficial for the development of both oil hydrolysis industry and biodegradable materials manufacturing industry. In order to construct an L-lactate producer, we first cloned a coding region of gene BcoaLDH encoding an L-lactate dehydrogenase from Bacillus coagulans CICIM B1821 and the promoter sequence (P(ldhA)) of the D-lactate dehydrogenase (LdhA) from Escherichia coli CICIM B0013. Then we assembled these two DNA fragments in vitro and yielded an expression cassette, P(ldhA)-BcoaLDH.

[Temperature-switched high-efficiency D-lactate production from glycerol].

Glycerol from oil hydrolysis industry is being considered as one of the abundent raw materials for fermentation industry. In present study, the aerobic and anaerobic metabolism and growth properties on glycerol by Esherichia coli CICIM B0013-070, a D-lactate over-producing strain constructed previously, at different temperatures were investigated, followed by a novel fermentation process, named temperature-switched process, was established for D-lactate production from glycerol. Under the optimal condition, lactate yield was increased from 64.

Metabolic engineering of Escherichia coli: a sustainable industrial platform for bio-based chemical production.

In order to decrease carbon emissions and negative environmental impacts of various pollutants, more bulk and/or fine chemicals are produced by bioprocesses, replacing the traditional energy and fossil based intensive route. The Gram-negative rod-shaped bacterium, Escherichia coli has been studied extensively on a fundamental and applied level and has become a predominant host microorganism for industrial applications. Furthermore, metabolic engineering of E.

Genetically switched D-lactate production in Escherichia coli.

During a fermentation process, the formation of the desired product during the cell growth phase competes with the biomass for substrates or inhibits cell growth directly, which results in a decrease in production efficiency. A genetic switch is required to precisely separate growth from production and to simplify the fermentation process. The ldhA promoter, which encodes the fermentative D-lactate dehydrogenase (LDH) in the lactate producer Escherichia coli CICIM B0013-070 (ack-pta pps pflB dld poxB adhE frdA), was replaced with the λ p(R) and p(L) promoters (as a genetic switch) using genomic recombination and the thermo-controllable strain B0013-070B (B0013-070, ldhAp::kan-cI(ts)857-p(R)-p(L)), which could produce two-fold higher LDH activity at 42°C than the B0013-070 strain, was created.

Fine tuning the transcription of ldhA for D-lactate production.

Fine tuning of the key enzymes to moderate rather than high expression levels could overproduce the desired metabolic products without inhibiting cell growth. The aims of this investigation were to regulate rates of lactate production and cell growth in recombinant Escherichia coli through promoter engineering and to evaluate the transcriptional function of the upstream region of ldhA (encoding fermentative lactate dehydrogenase in E. coli).

Improvement of D-lactate productivity in recombinant Escherichia coli by coupling production with growth.

Coupling lactate fermentation with cell growth was investigated in shake-flask and bioreactor cultivation systems by increasing aeration to improve lactate productivity in Escherichia coli CICIM B0013-070 (ackA pta pps pflB dld poxB adhE frdA). In shake-flasks, cells reached 1 g dry wt/l then, cultivated at 100 rpm and 42°C, achieved a twofold higher productivity of lactic acid compared to aerobic and O(2)-limited two-phase fermentation. The cells in the bioreactor yielded an overall volumetric productivity of 5.

[Elimination of succinate and acetate synthesis in recombinant Escherichia coli for D-lactate production].

When Escherichia coli CICIM B0013-030 (B0013, ack-pta, pps, pflB) was used for D-lactate production, succinate and acetate were the main byproducts (as much as 11.9 and 7.1% the amount of lactate respectively).

Evaluation of genetic manipulation strategies on D-lactate production by Escherichia coli.

In order to rationally manipulate the cellular metabolism of Escherichia coli for D: -lactate production, single-gene and multiple-gene deletions with mutations in acetate kinase (ackA), phosphotransacetylase (pta), phosphoenolpyruvate synthase (pps), pyruvate formate lyase (pflB), FAD-binding D-lactate dehydrogenase (dld), pyruvate oxidase (poxB), alcohol dehydrogenase (adhE), and fumarate reductase (frdA) were tested for their effects in two-phase fermentations (aerobic growth and oxygen-limited production). Lactate yield and productivity could be improved by single-gene deletions of ackA, pta, pflB, dld, poxB, and frdA in the wild type E. coli strain but were unfavorably affected by deletions of pps and adhE.