Cofactor engineering improved CALB production in Pichia pastoris through heterologous expression of NADH oxidase and adenylate kinase

Charumathi, Jayachandran; Balakumaran, Palanisamy Athiyaman; Meenakshisundaram, S.

doi:10.1371/journal.pone.0181370

Cited by 17 publications

(8 citation statements)

References 41 publications

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“… One of the key factors in the two-step cascade synthesis may include discovery of suitable enzymes and cofactor recycling. NAD(P) + -dependent ADH and ALDH − can be used repeatedly by the cofactor regeneration system through the coupling of the NAD(P)H-oxidases (NOXs). , We have conducted a literature survey and screening of novel enzymes based on sequence and structure information. Not only ADHs and ALDHs but also NOXs including old yellow enzymes (NAD(P)H flavin oxidoreductases, NFOs) from different species were examined in detail (Tables S6–S8).…”

Section: Resultsmentioning

confidence: 99%

“…The ADH from Kangiella koreensis (Kk), ALDH from Rodococcus erythropolis (Re), and NFO from Deinococcus radiodurans , which were expressed in E. coli as Biocatalyst II , were selected because of their high activity in the production of undecanedioic acid ( 7 ) from 5 (Figure a, Table S9). The productivity of Biocatalyst II for 30 min was 9.2-fold higher than that of E. coli expressing AlkJ, which has been used as a biocatalyst for producing 7 .…”

Section: Resultsmentioning

confidence: 99%

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Multilayer Engineering of Enzyme Cascade Catalysis for One-Pot Preparation of Nylon Monomers from Renewable Fatty Acids

et al. 2020

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Enzyme cascade catalysis has critical problems in obtaining the high concentrations of products, such as the low stabilities and activities of biocatalysts and the inhibition by hydrophobic reactants at high concentrations to biocatalysts. Here, we performed multilayer engineering of enzyme cascade catalysis to produce C11 nylon monomers at commercially viable concentrations from ricinoleic acid. The catalysis was driven by engineered Escherichia coli-based whole-cell biocatalysts and cell-free enzymes (i.e., lipases). Stabilities and activities of the biocatalysts were improved by engineering the bottleneck enzyme Baeyer− Villiger monooxygenase and introducing a cofactor regeneration system. The inhibitory effects of the byproducts n-heptanoic acid and pyruvate on the cascade enzymes were overcome, and the products were simply recovered in situ by engineering of reactions with the addition of an adsorbent resin. We obtained 248 mM undecanedioic acid or 232 mM 11-aminoundecanoic acid as a nylon monomer from 300 mM ricinoleic acid by multilayer engineering. The concentration was 500-and 640-fold higher than those produced by nonengineering, respectively. We have also simply isolated the C11 nylon monomers via solvent extraction to a rather high purity. This study will contribute to the industrial enzyme cascade synthesis of nylon monomers in an environmentallyfriendly route from renewable biomass.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Multilayer Engineering of Enzyme Cascade Catalysis for One-Pot Preparation of Nylon Monomers from Renewable Fatty Acids

et al. 2020

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“…ADH and ALDH in recombinant cells converted 15‐hydroxypentadecanoic acid and NAD(P) + to pentadecanedioic acid and NAD(P)H. Therefore, the regeneration of NAD(P) + as a co‐factor is required for increased conversion (Geueke et al, 2003; Jayachandran et al, 2017; Uppada et al, 2014) (Figure 2). For co‐factor regeneration, NOX genes from A. thaliana (AtNOX), C. albicans (CaNOX), L. brevis (LbNOX), L. esculentum (LeNOX), and T. denticola (TdNOX), and NFO from D. radiodurans (DrNFO) were introduced to E. coli cells expressing KkADH and GkALDH.…”

Section: Resultsmentioning

confidence: 99%

Pentadecanedioic acid production from 15‐hydroxypentadecanoic acid using an engineered biocatalyst with a co‐factor regeneration system

Shin

Kang

Lee

et al. 2022

J Americ Oil Chem Soc

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α,ω-Dicarboxylic acids are valuable precursors in various chemical industries and have recently been produced using biotechnological methods to overcome the limitations of chemical synthesis. Nonanedioic acid, decanedioic acid, undecanedioic acid, and dodecanedioic acid have been produced at high concentrations from ω-hydroxycarboxylic acids using engineered biocatalysts. However, no study has been attempted on the efficient production of pentadecanedioic acid. Here, the production of pentadecanedioic acid from 15-hydroxypentadecanoic acid was carried out with alcohol dehydrogenases (ADHs), aldehyde dehydrogenases (ALDHs), and NAD(P)H oxidases, including NAD(P)H flavin oxidoreductase (NFO), that used a co-factor regeneration system, derived from several species and expressed in Escherichia coli. Among the enzymes, Kangiella koreensis ADH (KkADH), Geobacillus kaustophilus ALDH (GkALDH), and Deinococcus radiodurans NFO (DrNFO) were selected because they had the highest activity. E. coli expressing pRSF-DrNFO and pACYC-KkADH/GkALDH as the best distribution of three genes in two plasmids was used as a biocatalyst to produce pentadecanedioic acid. The optimal conditions for producing pentadecanedioic acid from 15-hydroxypentadecanoic acid by the biocatalyst were pH 8.0, 35 C, 5% (v/v) methanol, 40 g L À1 cells, and 60 mM 15-hydroxypentadecanoic acid with agitation at 250 rpm. Under these optimized conditions, 57.4 mM pentadecanedioic acid was produced after 3 h, with a molar yield of 95.6% and a productivity of 19.1 mM h À1 . The molar yield and concentration of pentadecanedioic acid showed the highest values among the reported biotechnological production of pentadecanedioic acid.

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“…The largest increase in 3-HP production (36%) was achieved by targeting the gene encoding adenylate kinase 1, ADK1 #15 (TSS −761), which reversibly converts two ADPs to AMP and ATP. This gRNA increased 3-HP yields by 36% compared to the control strain (Figure ) (Table S4), and led to a downregulating effect on the activity of the ADK1 promoter coupled to GFP (Figure S3). Upon closer inspection, we noticed that the gRNA also binds downstream of the HTA1 coding sequence.…”

Section: Resultsmentioning

confidence: 99%

Model-Assisted Fine-Tuning of Central Carbon Metabolism in Yeast through dCas9-Based Regulation

et al. 2019

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Engineering Saccharomyces cerevisiae for industrial-scale production of valuable chemicals involves extensive modulation of its metabolism. Here, we identified novel gene expression fine-tuning set-ups to enhance endogenous metabolic fluxes toward increasing levels of acetyl-CoA and malonyl-CoA. dCas9-based transcriptional regulation was combined together with a malonyl-CoA responsive intracellular biosensor to select for beneficial set-ups. The candidate genes for screening were predicted using a genome-scale metabolic model, and a gRNA library targeting a total of 168 selected genes was designed. After multiple rounds of fluorescence-activated cell sorting and library sequencing, the gRNAs that were functional and increased flux toward malonyl-CoA were assessed for their efficiency to enhance 3-hydroxypropionic acid (3-HP) production. 3-HP production was significantly improved upon fine-tuning genes involved in providing malonyl-CoA precursors, cofactor supply, as well as chromatin remodeling.

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Cofactor engineering improved CALB production in Pichia pastoris through heterologous expression of NADH oxidase and adenylate kinase

Cited by 17 publications

References 41 publications

Multilayer Engineering of Enzyme Cascade Catalysis for One-Pot Preparation of Nylon Monomers from Renewable Fatty Acids

Multilayer Engineering of Enzyme Cascade Catalysis for One-Pot Preparation of Nylon Monomers from Renewable Fatty Acids

Pentadecanedioic acid production from 15‐hydroxypentadecanoic acid using an engineered biocatalyst with a co‐factor regeneration system

Model-Assisted Fine-Tuning of Central Carbon Metabolism in Yeast through dCas9-Based Regulation

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