Engineering Lactococcus lactis as a multi-stress tolerant biosynthetic chassis by deleting the prophage-related fragment

Qiao, Wanjin; Qiao, Yu; Liu, Fulu; Zhang, Yating; Li, Ran; Wu, Zhenzhou; Xu, Haijin; Saris, Per E. J.; Qiao, Mingqiang

doi:10.1186/s12934-020-01487-x

Cited by 11 publications

(13 citation statements)

References 66 publications

(68 reference statements)

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“…As suggested with the glpK and cyaA ALE mutations from the glycerol carbon-source case study, designs could also result from a combined set of compatible variants, where each variant optimizes for a separate stress. A strain's environment is naturally composed of multiple conditions, therefore compatible optimizations would be valuable in addressing multi-stress circumstances such as those of industrial scale fermentation (physical, chemical, and biological) [41][42][43] .…”

Section: Discussionmentioning

confidence: 99%

Data-Driven Strain Design Using Aggregated Adaptive Laboratory Evolution Mutational Data

Phaneuf¹,

Zielinski

Yurkovich³

et al. 2021

Preprint

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Microbes are being engineered for an increasingly large and diverse set of applications. However, the designing of microbial genomes remains challenging due to the general complexity of biological system. Adaptive Laboratory Evolution (ALE) leverages nature's problem-solving processes to generate optimized genotypes currently inaccessible to rational methods. The large amount of public ALE data now represents a new opportunity for data-driven strain design. This study presents a novel and first of its kind meta-analysis workflow to derive data-driven strain designs from aggregate ALE mutational data using rich mutation annotations, statistical and structural biology methods. The mutational dataset consolidated and utilized in this study contained 63 Escherichia coli K-12 MG1655 based ALE experiments, described by 93 unique environmental conditions, 357 independent evolutions, and 13,957 observed mutations. High-level trends across the entire dataset were established and revealed that ALE-derived strain designs will largely be gene-centric, as opposed to non-coding, and a relatively small number of variants (approx. 4) can significantly alter cellular states and provide benefits which range from an increase in fitness to a complete necessity for survival. Three novel experimentally validated designs relevant to metabolic engineering applications are presented as use cases for the workflow. Specifically, these designs increased growth rates with glycerol as a carbon source through a point mutation to glpK and a truncation to cyaA or increased tolerance to toxic levels of isobutyric acid through a pykF truncation. These results demonstrate how strain designs can be extracted from aggregated ALE data to enhance strain design efforts.

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Section: Discussionmentioning

confidence: 99%

Data-Driven Strain Design Using Aggregated Adaptive Laboratory Evolution Mutational Data

Phaneuf¹,

Zielinski

Yurkovich³

et al. 2021

Preprint

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“…The vectors for the deletion of nonessential fragments were constructed as described in our previous work [12]. Briefly, two fragments of the flanking region of nonessential fragment L1 were amplified by PCR with a proof-reading polymerase (Takara, Dalian, China), and the N8 chromosome was used as template.…”

Section: Methods Of Genome Streamliningmentioning

confidence: 99%

“…Cloning host; F-ϕ80lacZ∆M15endA1 recA1 endA1 hsdR17 (rK-mK+) supE44 thi-1 gyrA 96 relA1 ∆(lacZYA-argF)U169 deoR λ- [22] E. coli DH5α-up-down (L1) Cm r , Em r , E. coli DH5α derivative containing the whole plasmid pNZ5319∆L1 [12] pNZTS-Cre Em r , cre gene cloned at the EcoRI and HindIII sites (cat gene deletion vector) [20] pNZ5319∆L1 Cm r , Em r , upstream and downstream homology arm of L1 amplified from L. lactis N8 genome cloned into pNZ5319 [12] pNZ5319∆L2 Cm r , Em r , upstream and downstream homology arm of L2…”

Section: E Coli Dh5αmentioning

confidence: 99%

“…Library construction and sequencing were performed at the Beijing Novogene Bioinformatics Technology Co., Ltd. Quality control of paired-end reads was performed using in-house program. Then, data processing, reads mapping and SV (structural variation) analysis were conducted as we described previously [12].…”

Section: Resequencingmentioning

confidence: 99%

“…Researchers have tried to increase the nisin production of N8 through various methods, such as: (1) by improving the nisin immunity of the producer strain [12]; (2) by optimizing the culture conditions to achieve a high nisin yield of the producer strain [6]; (3) by using Mu transposition technology to build a library to screen genes that affect nisin production and nisin immunity [13,14].…”

Section: Introductionmentioning

confidence: 99%

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Genomic Features and Construction of Streamlined Genome Chassis of Nisin Z Producer Lactococcus lactis N8

et al. 2021

Self Cite

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Lactococcus lactis is a commonly used fermenting bacteria in cheese, beverages and meat products. Due to the lack of simplified chassis strains, it has not been widely used in the fields of synthetic biology. Thus, the construction of lactic acid bacteria chassis strains becomes more and more important. In this study, we performed whole genome sequencing, annotation and analysis of L. lactis N8. Based on the genome analysis, we found that L. lactis N8 contains two large plasmids, and the function prediction of the plasmids shows that some regions are related to carbohydrate transport/metabolism, multi-stress resistance and amino acid uptake. L. lactis N8 contains a total of seven prophage-related fragments and twelve genomic islands. A gene cluster encoding a hybrid NRPS–PKS system that was found in L. lactis N8 reveals that the strain has the potential to synthesize novel secondary metabolites. Furthermore, we have constructed a simplified genome chassis of L. lactis N8 and achieved the largest amount of deletion of L. lactis so far. Taken together, the present study offers further insights into the function and potential role of L. lactis N8 as a model strain of lactic acid bacteria and lays the foundation for its application in the field of synthetic biology.

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Comparative transcriptome analysis reveals the contribution of amino acid transporters to acid tolerance in Lactococcus lactis

Zhu,

Zhang,

Yang

et al. 2024

Food Frontiers

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Amino acid transporters are promising targets for engineering acid‐tolerant strains of Lactococcus lactis. However, the simple overexpression of transporters alone is insufficient to achieve a highly acid‐resistant phenotype. This study investigated the effects of amino acid transporters on the acid‐stress tolerance of L. lactis. Here, we first verified the contribution of amino acid transporters to acid tolerance by overexpressing the ctrA, glnP, and glnQ genes in L. lactis. Transcriptome analysis revealed that most genes associated with specific amino acid transport, pyrimidine metabolism, and functional proteins, were upregulated in the overexpression strains. Among them, arginine biosynthesis (argG and argH), amino acid transport (yjeM and azlC), and pyrimidine metabolism were considered to be the most important regulatory mechanisms. Importantly, metabolite profiling revealed that the overexpression strains had higher intracellular levels of amino acids, particularly aspartate, glutamate, and arginine at low pH, as well as higher intracellular ATP levels, which was consistent with the corresponding gene‐expression levels. Finally, the simultaneous overexpression of glnP and glnQ led to a further improvement of acid tolerance in L. lactis. This study reveals the regulatory mechanisms of amino acid transporters, and provides a novel strategy for achieving higher acid tolerance via positive tandem expression approaches.

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Engineering Lactococcus lactis as a multi-stress tolerant biosynthetic chassis by deleting the prophage-related fragment

Cited by 11 publications

References 66 publications

Data-Driven Strain Design Using Aggregated Adaptive Laboratory Evolution Mutational Data

Data-Driven Strain Design Using Aggregated Adaptive Laboratory Evolution Mutational Data

Genomic Features and Construction of Streamlined Genome Chassis of Nisin Z Producer Lactococcus lactis N8

Comparative transcriptome analysis reveals the contribution of amino acid transporters to acid tolerance in Lactococcus lactis

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