Tailoring fructooligosaccharides composition with engineered Zymomonas mobilis ZM4

Braga, Adelaide; Gomes, Daniela; Rainha, João; Cardoso, Beatriz; Amorim, Cláudia; Silvério, Sara C.; Fernández-Lobato, María; Rodrigues, Joana Lúcia Lima Correia; Rodrigues, L. R.

doi:10.1007/s00253-022-12037-3

Cited by 8 publications

(5 citation statements)

References 48 publications

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“…The broad host range vector pBBR1MCS2 (Kovach et al., 1995 ) is frequently utilized in Z. mobilis (Braga et al., 2022 ; Herrera et al., 2021 ; Liu et al., 2020 ). pGB‐MPI‐068 carries the rep , oriV and mob elements of pBBR1MCS2 and was further modified to create acceptors fitting the Zymo‐Parts system.…”

Section: Resultsmentioning

confidence: 99%

Construction and comparison of different vehicles for heterologous gene expression in Zymomonas mobilis

Behrendt,

Vlachonikolou,

Tietgens

et al. 2024

Microbial Biotechnology

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Zymomonas mobilis has the potential to be an optimal chassis for the production of bulk chemicals derived from pyruvate. However, a lack of available standardized and characterized genetic tools hinders both efficient engineering of Z. mobilis and progress in basic research on this organism. In this study, a series of different shuttle vectors were constructed based on the replication mechanisms of the native Z. mobilis plasmids pZMO1, pZMOB04, pZMOB05, pZMOB06, pZMO7 and p29191_2 and on the broad host range replication origin of pBBR1. These plasmids as well as genomic integration sites were characterized for efficiency of heterologous gene expression, stability without selection and compatibility. We were able to show that a wide range of expression levels could be achieved by using different plasmid replicons. The expression levels of the constructs were consistent with the relative copy numbers, as determined by quantitative PCR. In addition, most plasmids are compatible and could be combined. To avoid plasmid loss, antibiotic selection is required for all plasmids except the pZMO7‐based plasmid, which is stable also without selection pressure. Stable expression of reporter genes without the need for selection was also achieved by genomic integration. All modules were adapted to the modular cloning toolbox Zymo‐Parts, allowing easy reuse and combination of elements. This work provides an overview of heterologous gene expression in Z. mobilis and adds a rich set of standardized genetic elements to an efficient cloning system, laying the foundation for future engineering and research in this area.

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Section: Resultsmentioning

confidence: 99%

Construction and comparison of different vehicles for heterologous gene expression in Zymomonas mobilis

Behrendt,

Vlachonikolou,

Tietgens

et al. 2024

Microbial Biotechnology

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“…The gene 0917 encodes β-fructofuranosidase [EC 3.2.1.26], and the gene 1378 encodes levansucrase [EC 2.4.1.10]. These two genes are important in sucrose catabolism ( Braga et al, 2022 ), and their downstream genes are either downregulated or of very low abundance. These results indicated that the transcription of 0917 and 1378 genes was significantly downregulated due to the decreased activity of catabolic sucrose in CR5301, caused by the addition of RC.…”

Section: Resultsmentioning

confidence: 99%

Integrated metabolite profiling and transcriptome analysis unraveling mechanism of RC catabolism in Paenarthrobacter ilicis CR5301

Li¹,

Sun²,

Cao³

et al. 2023

Front. Microbiol.

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Steviol glycosides are ideal sweeteners that are widely used in food, medicine, and cosmetics. Rebaudioside C (RC) is considered to be the third most abundant steviol glycoside, which has a bitter aftertaste that limits its application. Hydrolysis of RC to generate other bioactive steviol glycosides is an effective way to promote its additional utilization. In our previous study, a bacterium Paenarthrobacter ilicis CR5301 was isolated and identified for hydrolyzing RC with high efficiency. Herein, the expression profiles of P. ilicis CR5301 in the deletion and presence of RC were investigated by RNA-seq. The RC metabolites were identified by high-performance liquid chromatography and ultra-performance liquid chromatography-triple-time of flight mass spectrometry. Novel results were discovered in four aspects of research. First, the identification of metabolites revealed that four metabolites, namely, dulcoside A, dulcoside B, dulcoside A1, and steviol, were produced during RC metabolism. Second, RNA-seq analyses unraveled that 105 genes of P. ilicis CR5301 were significantly differentially expressed, and 7 pathways were significantly enriched. Third, independent RT-qPCR verified the accuracy and reliability of the RNA-seq results. Finally, a complete catabolic model of RC in P. ilicis CR5301 was proposed, and key genes were indicated in the RC catabolic metabolism by combining them with literature and sequence alignments. This study comprehensively unraveled the genes and pathways of RC catabolism in P. ilicis CR5301 at the transcriptional and metabolic levels. It provided new insights and evidence for understanding the mechanism of RC catabolism in bacteria. Key candidate genes may potentially contribute to the RC hydrolysis and preparation of other functional steviol glycosides in the future.

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“…Both strains could produce a mix of FOS with a high concentration of 6-kestose. However, Z. mobilis ZM4_pB1-sacB produced 73.4 ± 1.6 g/L of FOS [184].…”

Section: From Genetic Engineering To Synthetic Biology To Produce Fru...mentioning

confidence: 94%

Fructooligosaccharides (FOS) Production by Microorganisms with Fructosyltransferase Activity

Belmonte-Izquierdo,

Salomé-Abarca,

González-Hernández

et al. 2023

Fermentation

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Fructans are fructose-based polymers, defined as fructooligosaccharides (FOS), when they possess a short chain. These molecules are highly appreciated in the food and pharmaceutical international market and have an increasing demand worldwide, mainly for their prebiotic activity and, therefore, for all their health benefits to those who consume them constantly. Thus, new natural or alternative FOS production systems of industrial scale are needed. In this regard, microorganisms (prokaryotes and eukaryotes) have the potential to produce them through a wide and diverse number of enzymes with fructosyltransferase activity, which add a fructosyl group to sucrose or FOS molecules to elongate their chain. Microbial fructosyltransferases are preferred in the industry because of their high FOS production yields. Some of these enzymes include levansucrases, inulosucrases, and β-fructofuranosidases obtained and used through biotechnological tools to enhance their fructosyltransferase activity. In addition, characterizing new microorganisms with fructosyltransferase activity and modifying them could help to increase the production of FOS with a specific degree of polymerization and reduce the FOS production time, thus easing FOS obtention. Therefore, the aim of this review is to compile, discuss, and propose new perspectives about the microbial potential for FOS production through enzymes with fructosyltransferase activity and describe the modulation of FOS production yields by exogenous stimuli and endogenous modifications.

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Tailoring fructooligosaccharides composition with engineered Zymomonas mobilis ZM4

Cited by 8 publications

References 48 publications

Construction and comparison of different vehicles for heterologous gene expression in Zymomonas mobilis

Construction and comparison of different vehicles for heterologous gene expression in Zymomonas mobilis

Integrated metabolite profiling and transcriptome analysis unraveling mechanism of RC catabolism in Paenarthrobacter ilicis CR5301

Fructooligosaccharides (FOS) Production by Microorganisms with Fructosyltransferase Activity

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