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Biotechnology & Applied Microbiology
Lactococcus lactis is a food-grade, and generally recognized as safe, bacterium, which making it ideal for producing plasmid DNA (pDNA) or recombinant proteins for industrial or pharmaceutical applications. L. lactis harboring high copy numbers of plasmids for DNA vaccines production showed altered proteome profiles, when compared with a smaller copy number plasmid. For live mucosal vaccination applications, the cell-wall anchored antigens had shown more promising results, when compared with intracellular or secreted antigens. However, previous transcriptome and proteome studies demonstrated that engineering L. lactis to express membrane proteins, mainly with a eukaryotic background, increases the overall cellular burden. Genome engineering strategies could be used to knockout or overexpress the pinpointed genes, so as to increase the profitability of the process.
- recombinant protein production
- systems biology
- Lactococcus lactis
- cell factory
- transcriptome
- proteome
1. Introduction
Lactic Acid Bacteria (LAB) are a group of Gram-positive bacteria that produce lactic acid from the fermentation of sugars. They are chemo-organotrophic and non-spore forming bacteria, which can be found in fermented food and beverages, in the intestinal and genital tract of human and animals, and on plants [1]. Lactococcus lactis is a non-pathogenic LAB with a wide applicability in industry, especially as a starter for cheese fermentation. This species could also be found in the gastrointestinal tract of animals and on plants’ surfaces. L. lactis is also considered a research model in molecular biology and in the metabolism of LAB [2].
The major prokaryotic system used in the microbial cell factory is Escherichia coli, since it is able to produce very high levels of plasmids and recombinant proteins. Since several L. lactis strains are considered food-grade and Generally Recognized As Safe (GRAS), as opposed to E. coli, which produces highly immunogenic lipopolysaccharides (LPS), they are for use as cell factories for biotechnology applications [3,4]. Traditionally, L. lactis have been used for the production of food- or pharmaceutical-grade proteins or metabolites for industrial application [5]. Additionally, this species has also been used for the production of pharmaceutical-grade plasmid DNA for DNA vaccination [6,7], or as live vectors for the delivery of DNA, proteins or metabolites to mucosal surfaces [8], which contributed to a rise in the industrial (pharmaceutical, food, cosmetic and energy) relevance of this species [9].
With the availability of sequenced genomes and related data from different levels (e.g., genome, transcriptome, proteome, metabolome), together with genome editing tools, systems biology has emerged as an increasingly relevant field for the study of L. lactis [10,11]. The first step in gene expression is the transcription of the stored DNA genetic information into mRNA by RNA polymerase. There are several techniques and tools for a transcriptome analysis, such as DNA microarray, quantitative real-time PCR and high throughput sequencing (RNA-seq) [12]. The next step in gene expression is the translation of the mRNA into a protein. Proteomics can help to characterize the cell proteome at a given time by one of the following major groups of techniques: antibodies-based methods, such as ELISA (Enzyme-linked immunosorbent assay), immunoprecipitation, immune-electrophoresis and Western blot; gel-based methods, such as two-dimensional gel electrophoresis and differential gel electrophoresis; chromatographic methods, such as ion-exchange, size exclusion and affinity chromatography; analytical, functional and reverse phase microarrays; mass spectrometry methods; quantitative techniques, such as ICAT (isotope-coded affinity tag labeling), SILAC (stable isotope labeling with amino acids in cell culture) and iTRAQ (isobaric tag for relative and absolute quantitation); X-ray crystallography; and nuclear magnetic-resonance spectroscopy [13].
Herein intends to critically analyze the most recent studies dealing with the effect of overproducing recombinant proteins in the transcriptome and proteome of L. lactis. The detailed outcomes of the production of cytoplasmic and membrane-bound proteins in L. lactis transcriptome and proteome will also be addressed. This information can be used to design and engineer optimized strains for recombinant protein production, enhancing the value of L. lactis as a cell factory, by the overexpression of a rate-limiting gene or the deletion of a disadvantageous gene. The transcriptomic and proteomic analysis could also identify the best (or worse) producing clones, as well as the rate-limiting steps in relevant pathways. At a bio-process level, transcriptome and proteome studies can help to define the feeding strategy and optimize the culture conditions (e.g., temperature, pH, aeration) [12]. The cell response in diverse environments under different stresses could also provide important insights into how L. lactis cope with heterologous protein expression.
2. Transcriptome and Proteome Profiles of L. lactis in Response to Natural Stresses
An analysis of the transcriptome and proteome of L. lactis when exposed to a low pH and to high levels of lactate and undissociated lactic acid is relevant for any protein-production setting, since these conditions result from the natural bacterial growth and fermentation metabolism. Wu et al. [24] observed that the acid and lactate stresses inhibited the carbohydrate and energy metabolisms and affected the cell growth probably due to the feedback inhibition from lactic acid, together with the up-regulation of the arginine deiminase pathway, as a way to maintain the stability of the intracellular pH. Several molecular chaperones and proteases were differentially regulated as a response to lactic acid stress, while the expression of DNA repair proteins (DnaA, DnaN and LigA) was down-regulated. With increasing concentrations of lactic acid, the expression of the cell wall genes changes concomitantly, with several up- and down-regulated genes, resulting in peptidoglycan hydrolysis and cell autolysis [24].
Wu et al. [25] analyzed several genes that were involved in L. lactis resistance to acid stress. A strain overexpressing ythA (PspC family transcriptional regulator) had a 3.2-fold higher survival rate in response to a pH 3.0 acid shock, when compared with the wild-type strain. A transcriptome analysis of the strain overexpressing ythA showed that it had an up-regulation of the genes involved in the biosynthesis of amino acids, pyrimidines and exopolysaccharides [25]. The overexpression of the genes arcB (amino acid metabolism) and malQ (carbon metabolism) resulted in higher survival rates when L. lactis was exposed to a pH 4.0 acid shock [26]. The authors also performed a high-throughput screening of mutant libraries generated by UV and chemical mutagenesis. The most acid-tolerant strain revealed that the carbohydrate, amino acid and fatty acids metabolisms were the most affected by the acid stress [26].
Although lactic-acid bacteria are widely used as starters for food and beverages fermentations, the strains used are usually wild-type, without any artificial genetic modifications. The LAB wild-type status could reduce the efficiency of introducing some desired modifications using molecular biology tools. For example, if the strain has active coding genes for endonucleases or extracellular nucleases [27], its transformation with a plasmid will be very difficult because plasmids would be degraded. Furthermore, if the goal is the generation of a strain to produce heterologous proteins, the production of proteases [27] from a wild-type strain is undesired. There are several synthetic biology tools that make it possible to re-design and optimize bacteria, by knocking out non-essential genes, or/and overexpressing others, in order to redirect LAB metabolism for high quality plasmid or protein production [28]. Information from transcriptome and proteome studies are of utmost importance to wisely choosing which genes to remove or to overexpress within L. lactis strains, in order to improve its pDNA and heterologous protein expression yields.
3. Conclusions
The information from the transcriptome and proteome studies in L. lactis provides important insight into methods that can be used to engineer the vectors and/or the strains when the objective is to produce a high quality and quantity of pDNA or recombinant protein for pharmaceutical/industrial applications. One of the most important characteristics to consider when selecting a vector is the type of replication origin, since it will influence the PCN. The PCN in turn affects the L. lactis transcriptome and proteome, and ultimately the pDNA and recombinant protein yield and quality. An average PCN vector, such as the pIL253-derived vectors (pAMβ1 replicon), allows for the preservation of the metabolism and integrity of the L. lactis cells, representing a good choice for obtaining acceptable amounts of pDNA and protein. Additionally, the promoter strength influences the amount of protein produced and, consequently, impacts the cell metabolism. An alternative could be to engineer a vector (i.e., change the origin of replication, change the promoter) using appropriate synthetic biology tools, for use in the proposed application. The modification of the plasmid vector can only increase its efficacy to a certain extent, making it necessary to also consider the strain modification. The data from how the production of recombinant proteins by L lactis affects its transcriptome and proteome could provide important information about the genes available for genome engineering (e.g., knockout or overexpression).
For live mucosal vaccination applications, one should account for the impact on the cell growth and metabolism, since the antigen of interest must be produced during a minimum period of time. The cell-wall anchored antigens, instead of intracellular or secreted ones, demonstrated more promising results in live mucosal vaccination studies. However, the transcriptome and proteome studies show that engineering L. lactis to express membrane proteins increases the overall cellular burden.
More studies are needed to investigate ways to engineer the L. lactis genome, and consequently, its transcriptome, proteome and metabolome, in order to overcome the hurdles experienced and increase its efficiency and profitability in different applications.
This entry is adapted from the peer-reviewed paper 10.3390/microorganisms10020267
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