Engineering microbial cell factories: Metabolic engineering of <i>Corynebacterium glutamicum</i> with a focus on non‐natural products

Heider, Sabine A. E.; Wendisch, Volker F.

doi:10.1002/biot.201400590

Cited by 104 publications

(51 citation statements)

References 184 publications

(235 reference statements)

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“…C orynebacterium glutamicum , a high-GC content, Gram-positive soil bacterium, is an important organism for the industrial production of amino acids123, and it has been engineered to produce a variety of compounds, including polymer subunits and biofuels24. Many approaches have been developed for introducing site-directed mutations into C. glutamicum 5.…”

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confidence: 99%

CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum

et al. 2017

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Corynebacterium glutamicum is an important industrial metabolite producer that is difficult to genetically engineer. Although the Streptococcus pyogenes (Sp) CRISPR-Cas9 system has been adapted for genome editing of multiple bacteria, it cannot be introduced into C. glutamicum. Here we report a Francisella novicida (Fn) CRISPR-Cpf1-based genome-editing method for C. glutamicum. CRISPR-Cpf1, combined with single-stranded DNA (ssDNA) recombineering, precisely introduces small changes into the bacterial genome at efficiencies of 86–100%. Large gene deletions and insertions are also obtained using an all-in-one plasmid consisting of FnCpf1, CRISPR RNA, and homologous arms. The two CRISPR-Cpf1-assisted systems enable N iterative rounds of genome editing in 3N+4 or 3N+2 days. A proof-of-concept, codon saturation mutagenesis at G149 of γ-glutamyl kinase relieves L-proline inhibition using Cpf1-assisted ssDNA recombineering. Thus, CRISPR-Cpf1-based genome editing provides a highly efficient tool for genetic engineering of Corynebacterium and other bacteria that cannot utilize the Sp CRISPR-Cas9 system.

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CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum

et al. 2017

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“…he Gram-positive Corynebacterium glutamicum is mostly known for its application in the industrial production of amino acids, mainly L-glutamate and L-lysine (1,2), and has become a versatile cell factory for the production of various commodity products (3)(4)(5). C. glutamicum utilizes a large variety of sugars and organic acids as sources of carbon and energy (6,7) and additionally has been genetically engineered for the utilization of alternative feedstocks such as starch, glycerol, xylose, glucuronic acid, and N-acetylglucosamine (8)(9)(10)(11)(12).…”

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Transcription of Sialic Acid Catabolism Genes in Corynebacterium glutamicum Is Subject to Catabolite Repression and Control by the Transcriptional Repressor NanR

et al. 2016

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Corynebacterium glutamicum metabolizes sialic acid (Neu5Ac) to fructose-6-phosphate (fructose-6P) via the consecutive activity of the sialic acid importer SiaEFGI, N-acetylneuraminic acid lyase (NanA), N-acetylmannosamine kinase (NanK), N-acetylmannosamine-6P epimerase (NanE), N-acetylglucosamine-6P deacetylase (NagA), and glucosamine-6P deaminase (NagB). Within the cluster of the three operons nagAB, nanAKE, and siaEFGI for Neu5Ac utilization a fourth operon is present, which comprises cg2936, encoding a GntR-type transcriptional regulator, here named NanR. Microarray studies and reporter gene assays showed that nagAB, nanAKE, siaEFGI, and nanR are repressed in wild-type (WT) C. glutamicum but highly induced in a ⌬nanR C. glutamicum mutant. Purified NanR was found to specifically bind to the nucleotide motifs A [AC]G[CT][AC]TGATGT C[AT][TG]ATGT[AC]TA located within the nagA-nanA and nanR-sialA intergenic regions. Binding of NanR to promoter regions was abolished in the presence of the Neu5Ac metabolism intermediates GlcNAc-6P and N-acetylmannosamine-6-phosphate (ManNAc-6P). We observed consecutive utilization of glucose and Neu5Ac as well as fructose and Neu5Ac by WT C. glutamicum, whereas the deletion mutant C. glutamicum ⌬nanR simultaneously consumed these sugars. Increased reporter gene activities for nagAB, nanAKE, and nanR were observed in cultivations of WT C. glutamicum with Neu5Ac as the sole substrate compared to cultivations when fructose was present. Taken together, our findings show that Neu5Ac metabolism in C. glutamicum is subject to catabolite repression, which involves control by the repressor NanR. IMPORTANCENeu5Ac utilization is currently regarded as a common trait of both pathogenic and commensal bacteria. Interestingly, the nonpathogenic soil bacterium C. glutamicum efficiently utilizes Neu5Ac as a substrate for growth. Expression of genes for Neu5Ac utilization in C. glutamicum is here shown to depend on the transcriptional regulator NanR, which is the first GntR-type regulator of Neu5Ac metabolism not to use Neu5Ac as effector but relies instead on the inducers GlcNAc-6P and ManNAc-6P. The identification of conserved NanR-binding sites in intergenic regions within the operons for Neu5Ac utilization in pathogenic Corynebacterium species indicates that the mechanism for the control of Neu5Ac catabolism in C. glutamicum by NanR as described in this work is probably conserved within this genus. The Gram-positive Corynebacterium glutamicum is mostly known for its application in the industrial production of amino acids, mainly L-glutamate and L-lysine (1, 2), and has become a versatile cell factory for the production of various commodity products (3-5). C. glutamicum utilizes a large variety of sugars and organic acids as sources of carbon and energy (6, 7) and additionally has been genetically engineered for the utilization of alternative feedstocks such as starch, glycerol, xylose, glucuronic acid, and N-acetylglucosamine (8-12). In contrast to many other bacterial species, C. glutamic...

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“…The reduced adhA RNA levels and the impaired growth with ethanol in the presence of GABA are commensurate with catabolite repression by GABA. Catabolite repression is an unusual phenomenon in C. glutamicum which is known for its ability to co-utilize a wide range of carbon sources (Blombach and Seibold 2010;Heider and Wendisch 2015). The non-growth substrate serine is co-utilized with glucose (Netzer et al 2004b), while the non-growth substrate methanol (Lessmeier et al 2013) is subject to glucose repression by RamA (Witthoff et al 2013).…”

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confidence: 99%

A new metabolic route for the production of gamma-aminobutyric acid by Corynebacterium glutamicum from glucose

Jorge

Leggewie²,

Wendisch

2016

Amino Acids

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Gamma-aminobutyric acid (GABA), a non-protein amino acid widespread in nature, is a component of pharmaceuticals, foods, and the biodegradable plastic polyamide 4. Corynebacterium glutamicum shows great potential for the production of GABA from glucose. GABA added to the growth medium hardly affected growth of C. glutamicum, since a half-inhibitory concentration of 1.1 M GABA was determined. As alternative to GABA production by glutamate decarboxylation, a new route for the production of GABA via putrescine was established in C. glutamicum. A putrescine-producing recombinant C. glutamicum strain was converted into a GABA producing strain by heterologous expression of putrescine transaminase (PatA) and gamma-aminobutyraldehyde dehydrogenase (PatD) genes from Escherichia coli. The resultant strain produced 5.3 ± 0.1 g L of GABA. GABA production was improved further by adjusting the concentration of nitrogen in the culture medium, by avoiding the formation of the by-product N-acetylputrescine and by deletion of the genes for GABA catabolism and GABA re-uptake. GABA accumulation by this strain was increased by 51 % to 8.0 ± 0.3 g L, and the volumetric productivity was increased to 0.31 g L h; the highest volumetric productivity reported so far for fermentative production of GABA from glucose in shake flasks was achieved.

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Engineering microbial cell factories: Metabolic engineering of Corynebacterium glutamicum with a focus on non‐natural products

Cited by 104 publications

References 184 publications

CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum

CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum

Transcription of Sialic Acid Catabolism Genes in Corynebacterium glutamicum Is Subject to Catabolite Repression and Control by the Transcriptional Repressor NanR

A new metabolic route for the production of gamma-aminobutyric acid by Corynebacterium glutamicum from glucose

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