Abstract:Lactobacillus casei is a potential cell factory for the production of enzymes and bioactive molecules using episomal plasmids, which suffer from genetic instability. While chromosomal integration strategies can provide genetic stability of recombinant proteins, low expression yields limit their application. To address this problem, we developed a two-step integration strategy in Lb. casei
by combination of the LCABL_13040-50-60 recombineering system (comprised of LCABL_1340, LCABL_13050, and LCABL_13060) with … Show more
“…After transformation of pLEISS-crtEBI into L. lactis NZ9000, the resulting strain NZ4 produced 0.59 mg/L lycopene (Figure 2A,B). To avoid the utilization of antibiotics for the maintenance of the plasmids, the expression cassette P nisA -crtEBI was integrated into the chromosome of L. lactis, resulting in 11 integrants with 1 ± 0.02 to 15 ± 1.02 copies of P nisA -crtEBI by Cre/loxP-mediated site-specific recombination, 27 and the lycopene levels of these integrants were 0.09 to 0.54 mg/L (Figure 2C,D). The lycopene production of the integrant NZ5 carrying 15 ± 1.02 copies of P nisA -crtEBI had no obvious difference compared with that of the strain NZ4 (P > 0.05), indicating that repetitive integration of genes into the chromosome made the lycopene production comparable to that of the episomal plasmid, agreeing with the previous results.…”
Lactococcus lactis is a food-grade
chassis for delivery of bioactive molecules to the intestinal mucosa in situ, while its ability to produce lycopene for detoxification
of reactive oxidative species (ROS) is not realized yet. Here, L. lactis NZ9000 was engineered to synthesize lycopene
by heterologous expression of a gene cluster crtEBI in plasmids or chromosomes, yielding the recombinant strains NZ4
and NZ5 with 0.59 and 0.54 mg/L lycopene production, respectively.
To reroute the pyruvate flux to lycopene, the main lactate dehydrogenase
and α-acetolactate synthase pathways were sequentially disrupted.
The resultant strains NZΔldh-1 and NZΔldhΔals-1
increased lycopene accumulation to 0.70 and 0.73 mg/L, respectively,
while their biomasses were reduced by 12.42% and the intracellular
NADH/NAD+ ratios increased by 3.05- and 2.10-fold. To increase
the biomasses of these engineered strains, aerobic respiration was
activated and tuned by the addition of exogenous heme and oxygen.
As a result, the engineered L. lactis strains partly recovered the growth and redox balance, yielding
the lycopene levels of 0.91–1.09 mg/L. The engineered L. lactis strain protected the intestinal epithelial
cells NCM460 against H2O2 challenge, with a
30.09% increase of cell survival and a 29.2% decrease of the intracellular
ROS level compared with strain NZ9000 treatment. In summary, this
work established the use of the engineered probiotic L. lactis for lycopene production and prospected
its potential in the prevention of intestinal oxidative damage.
“…After transformation of pLEISS-crtEBI into L. lactis NZ9000, the resulting strain NZ4 produced 0.59 mg/L lycopene (Figure 2A,B). To avoid the utilization of antibiotics for the maintenance of the plasmids, the expression cassette P nisA -crtEBI was integrated into the chromosome of L. lactis, resulting in 11 integrants with 1 ± 0.02 to 15 ± 1.02 copies of P nisA -crtEBI by Cre/loxP-mediated site-specific recombination, 27 and the lycopene levels of these integrants were 0.09 to 0.54 mg/L (Figure 2C,D). The lycopene production of the integrant NZ5 carrying 15 ± 1.02 copies of P nisA -crtEBI had no obvious difference compared with that of the strain NZ4 (P > 0.05), indicating that repetitive integration of genes into the chromosome made the lycopene production comparable to that of the episomal plasmid, agreeing with the previous results.…”
Lactococcus lactis is a food-grade
chassis for delivery of bioactive molecules to the intestinal mucosa in situ, while its ability to produce lycopene for detoxification
of reactive oxidative species (ROS) is not realized yet. Here, L. lactis NZ9000 was engineered to synthesize lycopene
by heterologous expression of a gene cluster crtEBI in plasmids or chromosomes, yielding the recombinant strains NZ4
and NZ5 with 0.59 and 0.54 mg/L lycopene production, respectively.
To reroute the pyruvate flux to lycopene, the main lactate dehydrogenase
and α-acetolactate synthase pathways were sequentially disrupted.
The resultant strains NZΔldh-1 and NZΔldhΔals-1
increased lycopene accumulation to 0.70 and 0.73 mg/L, respectively,
while their biomasses were reduced by 12.42% and the intracellular
NADH/NAD+ ratios increased by 3.05- and 2.10-fold. To increase
the biomasses of these engineered strains, aerobic respiration was
activated and tuned by the addition of exogenous heme and oxygen.
As a result, the engineered L. lactis strains partly recovered the growth and redox balance, yielding
the lycopene levels of 0.91–1.09 mg/L. The engineered L. lactis strain protected the intestinal epithelial
cells NCM460 against H2O2 challenge, with a
30.09% increase of cell survival and a 29.2% decrease of the intracellular
ROS level compared with strain NZ9000 treatment. In summary, this
work established the use of the engineered probiotic L. lactis for lycopene production and prospected
its potential in the prevention of intestinal oxidative damage.
“…GFP and FaeG were stably expressed in the recombinant strains without supplementation of the culture with antibiotics, and the protein production was comparable to that of a plasmid-engineered strain (Fig. 3 ) [ 47 ]. Fig.…”
Section: Gene Knock-out and Knock-in Technologies In Labmentioning
Lactic acid bacteria (LAB) are a phylogenetically diverse group with the ability to convert soluble carbohydrates into lactic acid. Many LAB have a long history of safe use in fermented foods and are recognized as food-grade microorganisms. LAB are also natural inhabitants of the human intestinal tract and have beneficial effects on health. Considering these properties, LAB have potential applications as biotherapeutic vehicles to delivery cytokines, antigens and other medicinal molecules. In this review, we summarize the development of, and advances in, genome manipulation techniques for engineering LAB and the expected future development of such genetic tools. These methods are crucial for us to maximize the value of LAB. We also discuss applications of the genome-editing tools in enhancing probiotic characteristics and therapeutic functionalities of LAB.
“…97 Furthermore, Xin et al also found 3.7-fold improvement in expression of the gfp gene at different genomic sites in Lactobacillus casei. 98 In summary, optimization of genomic position provides a meaningful strategy for improving the expression level of integrated expression.…”
Section: Other Strategies For Regulation Of Expressionmentioning
confidence: 99%
“…Moreover, Bilyk et al found that the expression of the reporter gene and aranciamycin biosynthetic cluster varies up to 8-fold depending on its position on the chromosome in Streptomyces albus J1074 . Furthermore, Xin et al also found 3.7-fold improvement in expression of the gfp gene at different genomic sites in Lactobacillus casei . In summary, optimization of genomic position provides a meaningful strategy for improving the expression level of integrated expression.…”
Section: Versatile Strategies To Improve the Integrated
Expression Of...mentioning
Chromosomal
integration of exogenous genes is preferred for industrially
related fermentation, as plasmid-mediated fermentation leads to extra
metabolic burden and genetic instability. Moreover, with the development
and advancement of genome engineering and gene editing technologies,
inserting genes into chromosomes has become more convenient; integration
expression is extensively utilized in microorganisms for industrial
bioproduction and expected to become the trend of recombinant protein
expression. However, in actual research and application, it is important
to enhance the expression of heterologous genes at the host genome
level. Herein, we summarized the basic principles and characteristics
of genomic integration; furthermore, we highlighted strategies to
improve the expression of chromosomal integration of genes and pathways
in host strains from three aspects, including chassis cell optimization,
regulation of expression elements in gene expression cassettes, optimization
of gene dose level and integration sites on chromosomes. Moreover,
we reviewed and summarized the relevant studies on the application
of integrated expression in the exploration of gene function and the
various types of industrial microorganism production. Consequently,
this review would serve as a reference for the better application
of integrated expression.
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