Improved l-Leucine Production in Corynebacterium glutamicum by Optimizing the Aminotransferases

Feng, Liyan; Xu, Jian-Zhong; Zhang, Weiguo

doi:10.3390/molecules23092102

Cited by 23 publications

(11 citation statements)

References 41 publications

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“…This strain C. glutamicum MV-LeuF2 produced up to 181 mM L-leucine in the chemically defined medium along with precipitate of L-leucine after 72 h of the fed-batch fermentation [ 27 ]. Feng et al reported that a rational modification of aminotransferase activity improved L-leucine production through optimizing the aminotransferases [ 44 ]. L-leucine producing strain C. glutamicum WL-14, obtained by rational genetic engineering with mutant bacteria as the starting strain, exhibited the high L-leucine titer of 28.5 g L -1 [ 45 ].…”

Section: Resultsmentioning

confidence: 99%

Effect of fed-batch and chemostat cultivation processes of C. glutamicum CP for L-leucine production

et al. 2021

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Most of the current industrial processes for L-leucine production are based on fermentation, usually in fed-batch operation mode. Although the culture technology has advanced in recent decades, the process still has significant drawbacks. To solve these problems, we investigated the effects of chemostat culture conditions on the production of L-leucine by Corynebacterium glutamicum CP. The dilution rate, the nitrogen source, and the carbon-nitrogen ratio of the medium were optimized. With the addition of ammonium acetate to the chemostat medium, the initial C/N ratio was adjusted to 57.6, and the L-leucine titer reached the highest level at the optimal dilution rate of 0.04 h −1. Compared with fed-batch culture, the L-leucine titer was reduced (from 53.0 to 24.8 g L −1), but the yield from glucose was increased by 10.0% (from 0.30 to 0.33 mol mol −1) and productivity was increased by 58.3% (from 1.2 to 1.9 g L −1 h −1). Moreover, the titer of the by-product L-alanine was significantly reduced (from 8.9 to 0.8 g L −1). In addition, gene expression levels and activity of key enzymes in the synthesis of L-leucine and L-alanine were analyzed to explain the difference of production performance between chemostat culture and fed-batch culture. The results indicate that chemostat culture has great potential to increase the industrial production of L-leucine compared to current fed-batch approaches.

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Section: Resultsmentioning

confidence: 99%

Effect of fed-batch and chemostat cultivation processes of C. glutamicum CP for L-leucine production

et al. 2021

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“…Strains used in this study are listed in This study Batch shake-ask fermentation was carried out in 500 mL shake ask with 50 mL of fermentation medium, and was referred to the methods described by Feng et al (2018). Inoculum was from a seed culture with ∆OD 562 =0.45-0.50 (at a dilution of 25-fold), and the inoculation amount was 10%.…”

Section: Strains Growth Medium and Culture Conditionsmentioning

confidence: 99%

An NADPH-auxotrophic Corynebacterium glutamicum recombinant strain and used it to construct L-leucine high-yielding strain

Chen

Liu

Zhang

et al. 2022

Preprint

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The NADPH-regeneration enzymes in Corynebacterium glutamicum were inactivated to construct an NADPH-auxotrophic C. glutamicum strain by gene knockout and gene replacement. The resultant NADPH-auxotrophic C. glutamicum XL-1 ΔZMICg::ISm (i.e., strain Leu-1) grew well in the basic medium only with gluconate as carbon source. Replacement of the native glyceraldehyde 3-phosphate dehydrogenase (NAD-GapDHCg) by NADP-GapDHCa from Clostridium acetobutylicum is an effective strategy for producing L-leucine in NADPH-prototrophic strain XL-1 and NADPH-auxotrophic strain Leu-1, whereas the L-leucine yield did not differ significantly between these strains (14.1±1.8 g/L v.s. 16.2±1.1 g/L). Enhancing the carbon flux in biosynthetic pathway by recombinant expression plasmid pEC-ABNCE promoted L-leucine production, but the shortage NADPH supply limited the L-leucine yield. The mutated promoters of zwf and icdCg were introduced into C. glutamicum with NADP-GapDHCa and pEC-ABNCE increased L-leucine yield (32.8±1.7 g/L) and improved cell growth (OD562=41.3±4.2) because the resultant strain C. glutamicum XL-1 ΔMICg::ISm GCg::GCa Pzwf-D1 Picd-D2/pEC-ABNCE (i.e., strain Leu-9) exhibited the proper intracellular NADPH and NADH level. This is the first report of constructing an L-leucine high-yielding strain that reasonably supplies NADPH by optimizing the biosynthetic pathway of NADPH from an NADPH-auxotrophic strain.

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“…before alaT and replacing native promoters of ilvBNC and leuA with the strong promoter P tuf improved l -leucine accumulation to 26.8 g/L [ 43 ]. To address the generation of by-products due to the substrate diversity of TA, the ilvE gene encoding TA was knocked out, and aspartate aminotransferase (AspB) was overexpressed in C. glutamicum FA-1 [ 63 ]. That strain produced 82.6% more l -leucine (20.8 g/L) than the original strain, while l -valine production remained unaltered, indicating that AspB has higher specificity for the precursor of l -leucine than l -valine production [ 63 ].…”

Section: Regulation Of Metabolic Flux Of Bcaas Production Pathwaysmentioning

confidence: 99%

“…In addition, the substrate diversity of AHAIR, DHAD, and TA should be determined via mutagenesis or by replacing them with their isozymes with substrate specificity to realize the production of individual BCAAs. For example, transaminase TyrB from E. coli , which catalyzes only 2-ketoisocaproate to produce l -leucine, could replace the substrate-diverse transaminase TA in C. glutamicum to allocate more carbon source for the sole production of l -leucine [ 63 ].…”

Section: Regulation Of Metabolic Flux Of Bcaas Production Pathwaysmentioning

confidence: 99%

Metabolic engineering of Corynebacterium glutamicum for producing branched chain amino acids

Zheng

Chen

et al. 2021

Microb Cell Fact

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Background Branched chain amino acids (BCAAs) are widely applied in the food, pharmaceutical, and animal feed industries. Traditional chemical synthetic and enzymatic BCAAs production in vitro has been hampered by expensive raw materials, harsh reaction conditions, and environmental pollution. Microbial metabolic engineering has attracted considerable attention as an alternative method for BCAAs biosynthesis because it is environmentally friendly and delivers high yield. Main text Corynebacterium glutamicum (C. glutamicum) possesses clear genetic background and mature gene manipulation toolbox, and has been utilized as industrial host for producing BCAAs. Acetohydroxy acid synthase (AHAS) is a crucial enzyme in the BCAAs biosynthetic pathway of C. glutamicum, but feedback inhibition is a disadvantage. We therefore reviewed AHAS modifications that relieve feedback inhibition and then investigated the importance of AHAS modifications in regulating production ratios of three BCAAs. We have comprehensively summarized and discussed metabolic engineering strategies to promote BCAAs synthesis in C. glutamicum and offer solutions to the barriers associated with BCAAs biosynthesis. We also considered the future applications of strains that could produce abundant amounts of BCAAs. Conclusions Branched chain amino acids have been synthesized by engineering the metabolism of C. glutamicum. Future investigations should focus on the feedback inhibition and/or transcription attenuation mechanisms of crucial enzymes. Enzymes with substrate specificity should be developed and applied to the production of individual BCAAs. The strategies used to construct strains producing BCAAs provide guidance for the biosynthesis of other high value-added compounds.

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Improved l-Leucine Production in Corynebacterium glutamicum by Optimizing the Aminotransferases

Cited by 23 publications

References 41 publications

Effect of fed-batch and chemostat cultivation processes of C. glutamicum CP for L-leucine production

Effect of fed-batch and chemostat cultivation processes of C. glutamicum CP for L-leucine production

An NADPH-auxotrophic Corynebacterium glutamicum recombinant strain and used it to construct L-leucine high-yielding strain

Metabolic engineering of Corynebacterium glutamicum for producing branched chain amino acids

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