Stepwise metabolic engineering of Gluconobacter oxydans WSH-003 for the direct production of 2-keto-l-gulonic acid from d-sorbitol

Gao, Lili; Hu, Yudong; Liu, Jie; Du, Guocheng; Zhou, Jingwen; Chen, Jian

doi:10.1016/j.ymben.2014.04.003

Cited by 68 publications

(51 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Previous studies indicated that enhancing the spatial proximity of enzymes through the creation of fusion constructs could regulate catalytic efficiency and enhance product synthesis in multi-enzyme reactions [25, 29, 30, 31]. The common and easiest approach to construct a fusion enzyme is to fuse the sequentially acting enzymes end to end by a linker peptide.…”

Section: Resultsmentioning

confidence: 99%

Carbonyl reductase identification and development of whole-cell biotransformation for highly efficient synthesis of (R)-[3,5-bis(trifluoromethyl)phenyl] ethanol

Chen

Deng

et al. 2016

Microb Cell Fact

View full text Add to dashboard Cite

Background(R)-[3,5-bis(trifluoromethyl)phenyl] ethanol [(R)-3,5-BTPE] is a valuable chiral intermediate for Aprepitant (Emend) and Fosaprepitant (Ivemend). Biocatalyzed asymmetric reduction is a preferred approach to synthesize highly optically active (R)-3,5-BTPE. However, the product concentration and productivity of reported (R)-3,5-BTPE synthetic processes remain unsatisfied.ResultsA NADPH-dependent carbonyl reductase from Lactobacillus kefir (LkCR) was discovered by genome mining for reduction of 3,5-bis(trifluoromethyl) acetophenone (3,5-BTAP) into (R)-3,5-BTPE with excellent enantioselectivity. In order to synthesize (R)-3,5-BTPE efficiently, LkCR was coexpressed with glucose dehydrogenase from Bacillus subtilis (BsGDH) for NADPH regeneration in Escherichia coli BL21 (DE3) cells, and the optimal recombinant strain produced 250.3 g/L (R)-3,5-BTPE with 99.9% ee but an unsatisfied productivity of 5.21 g/(L h). Then, four different linker peptides were used for the fusion expression of LkCR and BsGDH in E. coli to regulate catalytic efficiency of the enzymes and improved NADPH-recycling efficiency. Using the best strain (E. coli/pET-BsGDH-ER/K(10 nm)-LkCR), up to 297.3 g/L (R)-3,5-BTPE with enantiopurity >99.9% ee was produced via reduction of as much as 1.2 M of substrate with a 96.7% yield and productivity of 29.7 g/(L h).ConclusionsRecombinant E. coli/pET-BsGDH-ER/K(10 nm)-LkCR was developed for the bioreduction of 3,5-BTAP to (R)-3,5-BTPE, offered the best results in terms of high product concentration and productivity, demonstrating its great potential in industrial manufacturing of (R)-3,5-BTPE.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0585-5) contains supplementary material, which is available to authorized users.

show abstract

Section: Resultsmentioning

confidence: 99%

Carbonyl reductase identification and development of whole-cell biotransformation for highly efficient synthesis of (R)-[3,5-bis(trifluoromethyl)phenyl] ethanol

Chen

Deng

et al. 2016

Microb Cell Fact

View full text Add to dashboard Cite

show abstract

“…This was proven to be correct since induction of a pathway for PQQ synthesis resulted in a 20% increase in overall titer. A later refinement of the work in which the two pathway enzymes were incorporated as a fusion protein in Ketogulonigenium vulgare also resulted in a similar improvement in titer (Gao et al, 2014). …”

Section: Stimulating Cofactor Synthesis Can Enhance the Flux Of Synthmentioning

confidence: 97%

Cofactor Engineering for Enhancing the Flux of Metabolic Pathways

Akhtar

Jones

2014

Front. Bioeng. Biotechnol.

View full text Add to dashboard Cite

The manufacture of a diverse array of chemicals is now possible with biologically engineered strains, an approach that is greatly facilitated by the emergence of synthetic biology. This is principally achieved through pathway engineering in which enzyme activities are coordinated within a genetically amenable host to generate the product of interest. A great deal of attention is typically given to the quantitative levels of the enzymes with little regard to their overall qualitative states. This highly constrained approach fails to consider other factors that may be necessary for enzyme functionality. In particular, enzymes with physically bound cofactors, otherwise known as holoenzymes, require careful evaluation. Herein, we discuss the importance of cofactors for biocatalytic processes and show with empirical examples why the synthesis and integration of cofactors for the formation of holoenzymes warrant a great deal of attention within the context of pathway engineering.

show abstract

“…An Aminex HPX-87H column was used to perform HPLC separation at 35 • C with a flow rate of 0.5 mL/min and 5 mmol/L formic acid as the eluent. IT-TOF detection was performed with an ESI source in negative and positive ion mode at the followed conditions: detector voltage, 1.60 kV; nebulizing gas (N 2 ) flow, 1.5 L/min; drying gas (N 2 ) flow, 200 kPa; ion accumula-tion time, 30 ms; and scan range (m/ z), 100-300 for MS1 (Gao et al, 2014).…”

Section: Hplc and Liquid Chromatography Ion Trap Time-of-flight Mass mentioning

confidence: 99%

“…INTRODUCTION 2-Keto-L-gulonic acid (2-KLG) is the direct precursor for the industrial synthesis of vitamin C (Gao et al, 2014), and it can also make L-xylose, the precursor of L-xylitol . Both vitamin C and L-xylitol are important products used extensively in food, beverage and pharmaceutical industries (Zou et al, 2013;Ur-Rehman et al, 2015).…”

mentioning

confidence: 99%

High-Throughput Screening of a 2-Keto-L-Gulonic Acid-Producing Gluconobacter oxydans Strain Based on Related Dehydrogenases

Chen

Liu

Shan

et al. 2019

Front. Bioeng. Biotechnol.

Self Cite

View full text Add to dashboard Cite

High-throughput screening is a powerful tool for discovering strains in the natural environment that may be suitable for target production. Herein, a novel enzyme-based high-throughput screening method was developed for rapid screening of strains overproducing 2-keto-L-gulonic acid (2-KLG). The screening method detects changes in the fluorescence of reduced nicotinamide adenine dinucleotide (NADH) at 340 nm using a microplate reader when 2-KLG is degraded by 2-KLG reductase. In this research, three different 2-KLG reductases were expressed, purified, and studied. The 2-KLG reductase from Aspergillus niger were selected as the best appropriate reductase to establishment the method for its high activity below pH 7. Using the established method, and coupled with fluorescence-activated cell sorting, we achieved a high 2-KLG-producing strain of Gluconobacter oxydans WSH-004 from soil. When cultured with D-sorbitol as the substrate, the 2-KLG yield was 2.5 g/L from 50 g/L D-sorbitol without any side products. Compared with other reported screening methods, our enzyme-based method is more efficient and accurate for obtaining high-producing 2-KLG strains, and it is also convenient and cost-effective. The method is broadly applicable for screening keto acids and other products that can be oxidized via nicotinamide adenine dinucleotide (NAD + ) or nicotinamide adenine dinucleotide phosphate (NADP + ).

show abstract

Stepwise metabolic engineering of Gluconobacter oxydans WSH-003 for the direct production of 2-keto-l-gulonic acid from d-sorbitol

Cited by 68 publications

References 34 publications

Carbonyl reductase identification and development of whole-cell biotransformation for highly efficient synthesis of (R)-[3,5-bis(trifluoromethyl)phenyl] ethanol

Carbonyl reductase identification and development of whole-cell biotransformation for highly efficient synthesis of (R)-[3,5-bis(trifluoromethyl)phenyl] ethanol

Cofactor Engineering for Enhancing the Flux of Metabolic Pathways

High-Throughput Screening of a 2-Keto-L-Gulonic Acid-Producing Gluconobacter oxydans Strain Based on Related Dehydrogenases

Contact Info

Product

Resources

About