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CRISPRi-Library-Guided Target Identification for Engineering Carotenoid Production by Corynebacterium glutamicum.

ABSTRACT: Corynebacterium glutamicum is a prominent production host for various value-added compounds in white biotechnology. Gene repression by dCas9/clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) allows for the identification of target genes for metabolic engineering. In this study, a CRISPRi-based library for the repression of 74 genes of C. glutamicum was constructed. The chosen genes included genes encoding enzymes of glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle, regulatory genes, as well as genes of the methylerythritol phosphate and carotenoid biosynthesis pathways. As expected, CRISPRi-mediated repression of the carotenogenesis repressor gene crtR resulted in increased pigmentation and cellular content of the native carotenoid pigment decaprenoxanthin. CRISPRi screening identified 14 genes that affected decaprenoxanthin biosynthesis when repressed. Carotenoid biosynthesis was significantly decreased upon CRISPRi-mediated repression of 11 of these genes, while repression of 3 genes was beneficial for decaprenoxanthin production. Largely, but not in all cases, deletion of selected genes identified in the CRISPRi screen confirmed the pigmentation phenotypes obtained by CRISPRi. Notably, deletion of pgi as well as of gapA improved decaprenoxanthin levels 43-fold and 9-fold, respectively. The scope of the designed library to identify metabolic engineering targets, transfer of gene repression to stable gene deletion, and limitations of the approach were discussed.

SUBMITTER: Gottl VL

PROVIDER: S-EPMC8064071 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundCorynebacterium glutamicum contains the glycosylated C50 carotenoid decaprenoxanthin as yellow pigment. Starting from isopentenyl pyrophosphate, which is generated in the non-mevalonate pathway, decaprenoxanthin is synthesized via the intermediates farnesyl pyrophosphate, geranylgeranyl pyrophosphate, lycopene and flavuxanthin.ResultsHere, we showed that the genes of the carotenoid gene cluster crtE-cg0722-crtBIYeYfEb are co-transcribed and characterized defined gene deletion mutants. Gene deletion analysis revealed that crtI, crtEb, and crtYeYf, respectively, code for the only phytoene desaturase, lycopene elongase, and carotenoid C45/C50 ε-cyclase, respectively. However, the genome of C. glutamicum also encodes a second carotenoid gene cluster comprising crtB2I2-1/2 shown to be co-transcribed, as well. Ectopic expression of crtB2 could compensate for the lack of phytoene synthase CrtB in C. glutamicum ΔcrtB, thus, C. glutamicum possesses two functional phytoene synthases, namely CrtB and CrtB2. Genetic evidence for a crtI2-1/2 encoded phytoene desaturase could not be obtained since plasmid-borne expression of crtI2-1/2 did not compensate for the lack of phytoene desaturase CrtI in C. glutamicum ΔcrtI. The potential of C. glutamicum to overproduce carotenoids was estimated with lycopene as example. Deletion of the gene crtEb prevented conversion of lycopene to decaprenoxanthin and entailed accumulation of lycopene to 0.03 ± 0.01 mg/g cell dry weight (CDW). When the genes crtE, crtB and crtI for conversion of geranylgeranyl pyrophosphate to lycopene were overexpressed in C. glutamicum ΔcrtEb intensely red-pigmented cells and an 80 fold increased lycopene content of 2.4 ± 0.3 mg/g CDW were obtained.ConclusionC. glutamicum possesses a certain degree of redundancy in the biosynthesis of the C50 carotenoid decaprenoxanthin as it possesses two functional phytoene synthase genes. Already metabolic engineering of only the terminal reactions leading to lycopene resulted in considerable lycopene production indicating that C. glutamicum may serve as a potential host for carotenoid production.

Project description:BackgroundLignocellulose provides the only practical carbohydrates feedstock for sustainable bioproduction of hydrocarbons as future alternative of fossil fuels. Production of hydrocarbons from lignocellulose is achieved by a biorefinery process chain including pretreatment to breakdown the crystalline structure for cellulase-catalyzed hydrolysis, detoxification of inhibitory compounds generated during pretreatment, enzymatic hydrolysis to fermentable monosaccharide sugars, and fermentation to hydrocarbon products. The major barriers on fermentative production of hydrocarbons from lignocellulose include two aspects: one is the inherent stress of pretreatment-derived inhibitors on microbial cells, the other is the toxicity of hydrocarbons to cell membranes. The microbial cell factory should be tolerant to both inhibitor stress and hydrocarbons toxicity.ResultsCorynebacterium glutamicum was selected as the starting strain of hydrocarbons synthesis since it is well adapted to lignocellulose hydrolysate environment. The dual hydrocarbon synthesis pathways were constructed in an industrial C. glutamicum S9114 strain. The first pathway was the regular one in microalgae composed of fatty acyl-acyl carrier protein (fatty acyl-ACP) reductase (AAR) and aldehyde deformylating oxygenase (ADO) with fatty acyl-ACP as precursor. The second pathway was the direct decarboxylation of free fatty acid by fatty acid decarboxylase (OleT) using the rich fatty acids from the disruption of the transcriptional regulator fasR gene. The transmembrane transportation of hydrocarbon products was avoided by secretively expressing the fatty acid decarboxylase (OleT) to the extracellular space. The hydrocarbons generation from glucose reached 29.2 mg/L, in which the direct decarboxylation pathway contributed more than 70% of the total hydrocarbons generation, and the AAR-ADO pathway contributed the rest 30%.ConclusionThe dual hydrocarbon synthesis pathways (OleT and AAR-ADO pathways) were constructed in the inhibitors tolerant C. glutamicum S9114 strain for hydrocarbon production using lignocellulose feedstock as the starting feedstock. When corn stover was used for hydrocarbons production after dry acid pretreatment and biodetoxification, the hydrocarbons generation reached 16.0 mg/L. This study provided a new strategy for hydrocarbons synthesis using microbial cell factory suitable for lignocellulose feedstock.

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CRISPRi-Library-Guided Target Identification for Engineering Carotenoid Production by Corynebacterium glutamicum.

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