Single-step production of polyhydroxybutyrate from starch by using α-amylase cell-surface displaying system of Corynebacterium glutamicum

Song, Yue; Matsumoto, Ken’ichiro; Tanaka, Tsutomu; Kondo, Akihiko; Taguchi, Seiichi

doi:10.1016/j.jbiosc.2012.08.004

Cited by 37 publications

(17 citation statements)

References 22 publications

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“…As shown here for L-lysine, the strain engineered to generate NADPH through 22 glycolysis is likely to be an effective host for other NADPH-requiring compounds, such as other amino acids , commodity chemicals (Kind et al, 2011;Song et al, 2013), vitamins (Hüser et al, 2005;, fatty acids , and carotenoids (Heider et al, 2012). The results of the present study will pave the way for efficient production of these compounds.…”

Section: Discussionmentioning

confidence: 64%

l -Lysine production independent of the oxidative pentose phosphate pathway by Corynebacterium glutamicum with the Streptococcus mutans gapN gene

Takeno

Hori

Ohtani

et al. 2016

Metabolic Engineering

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We have recently developed a Corynebacterium glutamicum strain that generates NADPH via the glycolytic pathway by replacing endogenous NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GapA) with a nonphosphorylating NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (GapN) from Streptococcus mutans. Strain RE2, a suppressor mutant spontaneously isolated for its improved growth on glucose from the engineered strain, was proven to be a high-potential host for L-lysine production (Takeno et al., 2010). In this study, the suppressor mutation was identified to be a point mutation in rho encoding the transcription termination factor Rho. Strain RE2 still showed retarded growth despite the mutation rho696. Our strategy for reconciling improved growth with a high level of L-lysine production was to use GapA together with GapN only in the early growth phase, and subsequently shift this combination-type glycolysis to one that depends only on GapN in the rest of the growth phase. To achieve this, we expressed gapA under the 2 myo-inositol-inducible promoter of iolT1 encoding a myo-inositol transporter in strain RE2. The resulting strain RE2A iol was engineered into an L-lysine producer by introduction of a plasmid carrying the desensitized lysC, followed by examination for culture conditions with myo-inositol supplementation. We found that as a higher concentration of myo-inositol was added to the seed culture, the following fermentation period became shorter while maintaining a high level of L-lysine production. This finally reached a fermentation period comparable to that of the control GapA strain, and yielded a 1.5-fold higher production rate compared with strain RE2. The transcript level of gapA, as well as the GapA activity, in the early growth phase increased in proportion to the myo-inositol concentration and then fell to low levels in the subsequent growth phase, indicating that improved growth was a result of increased GapA activity, especially in the early growth phase. Moreover, blockade of the pentose phosphate pathway through a defect in glucose 6-phosphate dehydrogenase did not significantly affect L-lysine production in the engineered GapN strains, while a drastic decrease in L-lysine production was observed for the control GapA strain. Determination of the intracellular NADPH/NADP + ratios revealed that the ratios in the engineered strains were significantly higher than the ratio of the control GapA strain irrespective of the pentose phosphate pathway. These results demonstrate that our strain engineering strategy allows efficient L-lysine production independent of the oxidative pentose phosphate pathway.

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Section: Discussionmentioning

confidence: 64%

l -Lysine production independent of the oxidative pentose phosphate pathway by Corynebacterium glutamicum with the Streptococcus mutans gapN gene

Takeno

Hori

Ohtani

et al. 2016

Metabolic Engineering

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“…As another display model, we examined the α‐amylase from Streptococcus bovis 148, which can degrade starch to glucose, and whose display on bacterial cell surfaces can be used for the development of whole cell biocatalysts for the bioconversion of the starch biomass . We also constructed the two display platforms with full and short‐length NCgl1337 (pH36R‐NCgl1337F‐Amy and pH36R‐NCgl1337S‐Amy), and after flask cultivation, the production and display of amylase in each system were compared.…”

Section: Resultsmentioning

confidence: 99%

Development of a Potential Protein Display Platform in Corynebacterium glutamicum Using Mycolic Acid Layer Protein, NCgl1337, as an Anchoring Motif

Choi

Yim

Jeong

2017

Biotechnology Journal

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In the cell surface display, the choice of host cell and anchoring motif are the most crucial for the efficient display of passenger proteins. Corynebacterium glutamicum has mycolic acid layer in outer membrane and the use of protein in the mycolic acid layer as an anchoring motif can provide a potential platform for surface display in C. glutamicum. All 19 mycolic acid layer proteins of C. glutamicum are analyzed, and two proteins, NCgl0535 and NCgl1337, which have a signal peptide and predicted O-mycoloylation site, are selected as anchoring motifs candidates. Among them, NCgl1337, which shows better expression with higher display efficiency, is chosen as a potential anchoring motif. Two forms of the NCgl1337 anchoring motif, a full-length (1-324 amino acids) and a short-length (1-50 amino acids) containing only signal peptide and O-mycoloylation site, are constructed and their abilities for surface display are examined using two protein models, endoxylanase from Streptomyces coelicolor and α-amylase from Streptococcus bovis. For both model proteins, the short-length NCgl1337 anchoring motif exhibits higher yield of protein display on the surface of C. glutamicum than the full-length NCgl1337. Finally, with C. glutamicum displaying α-amylase, a batch fermentation is performed for the production of l-lysine from starch degradation, and a production of l-lysine as high as 10.8 ± 0.92 g L was achieved after 18 h of culture.

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“…Despite a broad product portfolio for C. glutamicum (15,17,18,19,21), lipids and their related compounds have not been intensively developed for production. In this study, we demonstrated for the first time that this organism has the capability of producing considerable amounts of fatty acids directly from sugar, thus expanding its product portfolio to lipids.…”

Section: Discussionmentioning

confidence: 99%

“…This bacterium has long been used for the industrial production of a variety of amino acids, including L-glutamic acid and L-lysine (15). It has also recently been developed as a production platform for various commodity chemicals (16,17,18), fuel alcohols (19,20), carotenoids (21), and heterologous proteins (22). However, there are no reports of fatty acid production by this bacterium, except for undesired production of acetate, a water-soluble short-chain fatty acid, as a by-product (23).…”

mentioning

confidence: 99%

Development of Fatty Acid-Producing Corynebacterium glutamicum Strains

Takeno

Takasaki

Urabayashi

et al. 2013

Appl Environ Microbiol

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bTo date, no information has been made available on the genetic traits that lead to increased carbon flow into the fatty acid biosynthetic pathway of Corynebacterium glutamicum. To develop basic technologies for engineering, we employed an approach that begins by isolating a fatty acid-secreting mutant without depending on mutagenic treatment. This was followed by genome analysis to characterize its genetic background. The selection of spontaneous mutants resistant to the palmitic acid ester surfactant Tween 40 resulted in the isolation of a desired mutant that produced oleic acid, suggesting that a single mutation would cause increased carbon flow down the pathway and subsequent excretion of the oversupplied fatty acid into the medium. Two additional rounds of selection of spontaneous cerulenin-resistant mutants led to increased production of the fatty acid in a stepwise manner. Whole-genome sequencing of the resulting best strain identified three specific mutations (fasR20, fasA63 up , and fasA2623). Allele-specific PCR analysis showed that the mutations arose in that order. Reconstitution experiments with these mutations revealed that only fasR20 gave rise to oleic acid production in the wild-type strain. The other two mutations contributed to an increase in oleic acid production. Deletion of fasR from the wild-type strain led to oleic acid production as well. Reverse transcription-quantitative PCR analysis revealed that the fasR20 mutation brought about upregulation of the fasA and fasB genes encoding fatty acid synthases IA and IB, respectively, by 1.31-fold ؎ 0.11-fold and 1.29-fold ؎ 0.12-fold, respectively, and of the accD1 gene encoding the ␤-subunit of acetyl-CoA carboxylase by 3.56-fold ؎ 0.97-fold. On the other hand, the fasA63 up mutation upregulated the fasA gene by 2.67-fold ؎ 0.16-fold. In flask cultivation with 1% glucose, the fasR20 fasA63 up fasA2623 triple mutant produced approximately 280 mg of fatty acids/liter, which consisted mainly of oleic acid (208 mg/liter) and palmitic acid (47 mg/liter).

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Single-step production of polyhydroxybutyrate from starch by using α-amylase cell-surface displaying system of Corynebacterium glutamicum

Cited by 37 publications

References 22 publications

l -Lysine production independent of the oxidative pentose phosphate pathway by Corynebacterium glutamicum with the Streptococcus mutans gapN gene

l -Lysine production independent of the oxidative pentose phosphate pathway by Corynebacterium glutamicum with the Streptococcus mutans gapN gene

Development of a Potential Protein Display Platform in Corynebacterium glutamicum Using Mycolic Acid Layer Protein, NCgl1337, as an Anchoring Motif

Development of Fatty Acid-Producing Corynebacterium glutamicum Strains

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