Engineering Of Lactic Acid Bacteria Research Articles

Novel Strategies for Efficient Production and Delivery of Live Biotherapeutics and Biotechnological Uses of Lactococcus lactis: The Lactic Acid Bacterium Model.

Lactic acid bacteria (LAB) are traditionally used in fermentation and food preservation processes and are recognized as safe for consumption. Recently, they have attracted attention due to their health-promoting properties; many species are already widely used as probiotics for treatment or prevention of various medical conditions, including inflammatory bowel diseases, infections, and autoimmune disorders. Some LAB, especially Lactococcus lactis, have been engineered as live vehicles for delivery of DNA vaccines and for production of therapeutic biomolecules. Here, we summarize work on engineering of LAB, with emphasis on the model LAB, L. lactis. We review the various expression systems for the production of heterologous proteins in Lactococcus spp. and its use as a live delivery system of DNA vaccines and for expression of biotherapeutics using the eukaryotic cell machinery. We have included examples of molecules produced by these expression platforms and their application in clinical disorders. We also present the CRISPR-Cas approach as a novel methodology for the development and optimization of food-grade expression of useful substances, and detail methods to improve DNA delivery by LAB to the gastrointestinal tract. Finally, we discuss perspectives for the development of medical applications of recombinant LABs involving animal model studies and human clinical trials, and we touch on the main safety issues that need to be taken into account so that bioengineered versions of these generally recognized as safe organisms will be considered acceptable for medical use.

Recent Advances in Synthetic Biology for the Engineering of Lactic Acid Bacteria

Lactic acid bacteria (LAB) have a long historical record of usage in many types of food-related bioprocesses, and as LAB have been found to have intrinsic beneficial effects on human health, interest in using LAB as a medical treatment has increased. Moreover, recent advances in synthetic biology have enabled the engineering of so-called “smart” bacteria with the native characteristics of LAB. With various synthetic parts for gene expression, genome editing, and genetic circuits, gene expression can be precisely regulated in LAB. Moreover, LAB have been successfully developed as potential hosts for therapeutics to detect and control specific diseases or to deliver drug molecules. In this review, we outline the recent development of synthetic parts and approaches for the engineering of LAB. We also discuss the potential application of synthetic biology for the utilization of LAB as living therapeutics.

Engineering of lactic acid bacteria for delivery of therapeutic proteins and peptides.

Lactic acid bacteria (LAB) have a long-term history of use in food industry and are becoming attractive for use in therapy on account of their safety, intrinsic beneficial health effects, and considerable biotechnological potential. The established systems for engineering are combined with novel approaches, such as CRISPR-Cas, to enable the use of LAB as vectors for delivery of various therapeutic molecules. The latter are either secreted or surface displayed and can be used for the treatment or prevention of numerous conditions: inflammatory bowel diseases, infections, autoimmune diseases, and even cancer. This review presents some recent data on engineering of LAB, with the emphasis on the most commonly used genera Lactococcus and Lactobacillus. Their use for the delivery of therapeutic proteins is discussed, while a special focus is given to the delivery of therapeutic peptides. Therapeutically relevant improvements of engineered LAB, such as containment systems, ability to visualize bacteria, or target specific host cells are also addressed. Future engineering of LAB for therapy will adopt the capabilities of synthetic biology, with first examples already emerging.

Cloning and Characterization of a Novel tuf Promoter from Lactococcus lactis subsp. lactis IL1403

Genetic engineering of lactic acid bacteria (LAB) requires a reliable gene expression system. Especially, a stable promoter is an important genetic element to induce gene expression in such a system. We report on a novel tuf promoter (Ptuf) of Lactococcus lactis subsp. lactis IL1403 that was screened and selected through analysis of previously published microarray data. Ptuf activity was examined and compared with three other known lactococcal promoters (PdnaJ, PpfkA, and Pusp45) using different bacteria as expression hosts. Each promoter was, respectively, fused to the promoterless and modified bmpB gene as a reporter, and we estimated promoter activity through BmpB expression. All promoters were active in IL1403, and Ptuf activity was strongest among them. The activity of each promoter differed by host bacteria (Lactobacillus plantarum Lb25, Lactobacillus reuteri ATCC23272, and Escherichia coli Top10F'). Ptuf had the highest activity in IL1403 when growth reached late log phase. The activity of each promoter correlated with the expression of each cognate gene in the microarray data (R (2) = 0.7186, P = 0.06968). This study revealed that novel food-grade promoters such as IL1403 Ptuf can be selected from microarray data for food-grade microorganisms and Ptuf can be used to develop a reliable gene expression system in L. lactis.

Immunomodulation by genetically engineered lactic acid bacteria

The taxonomically diverse lactic acid bacteria (LAB) are unified by their capability to produce lactic acid from carbohydrates by fermentation. The LAB Lactococcus (L.) lactis has been characterized into great detail and is increasingly used as a production host for heterologous proteins. L. lactis is a non-pathogenic and non-colonizing LAB species and can be efficiently engineered to produce proteins of viral, bacterial or eukaryotic origin, both intra- or extracellularly. Importantly, orally formulated L. lactis strains (ActoBiotics), engineered to synthesize and secrete therapeutic peptides and proteins in the gastrointestinal tract, are already in advanced stages of preclinical and clinical development. This review focuses on the genetic engineering of LAB in general and L. lactis in specific to secrete high-quality, correctly processed, bioactive molecules derived from a eukaryotic background. The therapeutic applications of these genetically modified strains are discussed, as well as the need for a sound environmental containment strategy, and a detailed review is presented on Lactococcus strains engineered to produce specific antigens, antibodies, cytokines and trefoil factors, with special regards to immunomodulation.

The relevance of genetic analysis to dairy bacteria: building upon our heritage

Lactic acid bacteria (LAB) are essential for the manufacture of fermented dairy products. Studies on the physiology, biochemistry and genetics of these microorganisms over the last century have contributed considerably to the improvement of fermentation processes and have resulted in better and safer products. Nevertheless, the potential of LAB is far from being maximized. The sophistication of biotechnologies and the availability of complete genome sequences have opened the door to the metabolic engineering of LAB. In this regard, the recent publication of the complete genome sequences of two Streptococcus thermophilus strains will provide a key tool to facilitate the genetic manipulation of this important dairy species.

Novel food-grade plasmid vector based on melibiose fermentation for the genetic engineering of Lactococcus lactis.

The alpha-galactosidase gene (aga) and a gene coding for a putative transcriptional regulator from the LacI/GalR family (galR) of Lactococcus raffinolactis ATCC 43920 were cloned and sequenced. When transferred into Lactococcus lactis and Pediococcus acidilactici strains, aga modified the sugar fermentation profile of the strains from melibiose negative (Mel(-)) to melibiose positive (Mel(+)). Analysis of galA mutants of L. lactis subsp. cremoris MG1363 indicated that the putative galactose permease GalA is also needed to obtain the Mel(+) phenotype. Consequently, GalA may also transport melibiose into this strain. We demonstrated that when aga was associated with the theta-type replicon of a natural L. lactis plasmid, it constituted the selectable marker of a cloning vector named pRAF800. Transcriptional analysis by reverse transcriptase PCR suggests that this vector is also suitable for gene expression. The alpha-galactosidase activity conferred by pRAF800 was monitored in an industrial strain grown in the presence of various carbon sources. The results indicated that the enzymatic activity was induced by galactose and melibiose, but not by glucose or lactose. The gene encoding the phage defense mechanism, AbiQ, was cloned into pRAF800, and the resulting clone (pRAF803) was transferred into an industrial L. lactis strain that became highly phage resistant. The measurements of various growth parameters indicated that cells were not affected by the presence of pRAF803. Moreover, the plasmid was highly stable in this strain even under starter production conditions. The L. raffinolactis aga gene represents the basis of a novel and convenient food-grade molecular tool for the genetic engineering of lactic acid bacteria.

Metabolic engineering of lactic acid bacteria, the combined approach: kinetic modelling, metabolic control and experimental analysis.

Everyone who has ever tried to radically change metabolic fluxes knows that it is often harder to determine which enzymes have to be modified than it is to actually implement these changes. In the more traditional genetic engineering approaches 'bottle-necks' are pinpointed using qualitative, intuitive approaches, but the alleviation of suspected 'rate-limiting' steps has not often been successful. Here the authors demonstrate that a model of pyruvate distribution in Lactococcus lactis based on enzyme kinetics in combination with metabolic control analysis clearly indicates the key control points in the flux to acetoin and diacetyl, important flavour compounds. The model presented here (available at http://jjj.biochem.sun.ac.za/wcfs.html) showed that the enzymes with the greatest effect on this flux resided outside the acetolactate synthase branch itself. Experiments confirmed the predictions of the model, i.e. knocking out lactate dehydrogenase and overexpressing NADH oxidase increased the flux through the acetolactate synthase branch from 0 to 75% of measured product formation rates.

Metabolic engineering of lactic acid bacteria for the production of nutraceuticals.

Lactic acid bacteria display a relatively simple and well-described metabolism where the sugar source is converted mainly to lactic acid. Here we will shortly describe metabolic engineering strategies on the level of sugar metabolism, that lead to either the efficient re-routing of the lactococcal sugar metabolism to nutritional end-products other than lactic acid such as L-alanine, several low-calorie sugars and oligosaccharides or to enhancement of sugar metabolism for complete removal of (undesirable) sugars from food materials. Moreover, we will review current metabolic engineering approaches that aim at increasing the flux through complex biosynthetic pathways, leading to the production of the B-vitamins folate and riboflavin. An overview of these metabolic engineering activities can be found on the website of the Nutra Cells 5th Framework EU-project (www.nutracells.com). Finally, the impact of the developments in the area of genomics and corresponding high-throughput technologies on nutraceutical production will be discussed.

Metabolic engineering of lactic acid bacteria: overview of the approaches and results of pathway rerouting involved in food fermentations.

Lactic acid bacteria such as Lactococcus lactis are the microorganisms of choice for performing metabolic engineering in relation to food fermentation. These bacteria are used extensively in food fermentations, they have a simple and therefore controllable metabolism and the molecular genetics of these food bacteria is well-developed. There have been recent successes in metabolic engineering in these lactic acid bacteria, including examples of changes in both primary metabolism (diacetyl and alanine) and secondary metabolism (exopolysaccharides and flavour).

Characterization of a novel Streptococcus thermophilus rolling-circle plasmid used for vector construction.

The complete nucleotide sequence of pER371, a native plasmid in Streptococcus thermophilus ST137, was determined. A putative open reading frame coding for a replication protein, Rep371, was identified. A characteristic promoter sequence and ribosome-binding site were found upstream of rep371. Rep371 (247 amino acid residues) does not show homology with RepA and RepS of the small S. thermophilus cryptic plasmids pST1-No.29 and pST1 respectively. The plus-origin sequence and Rep371 are highly homologous to the corresponding elements of the Staphylococcus aureus plasmids pC194 and pSK89. A novel 140-nucleotide palindromic minusorigin sequence, which is structurally similar but does not show sequence homology to the palA region of pC194, was identified in pER371. A palindromic sequence capable of forming a putative hairpin structure was identified and subsequently recognized as being highly conserved among several lactococcal rolling-circle plasmids. Cloning vectors derived from pER371 should provide valuable gene-delivery vehicles for the genetic engineering of lactic acid bacteria.

Characterization of pER371-based Streptococcus thermophilus-Escherichia coli shuttle vectors

Native plasmid of Streptococcus thermophilus ST137, pER371 (2.7 kb) linearized at various unique restriction sites was individually subcloned into Escherichia coli plasmid pUC19 to generate the pUER-series recombinants. A selection cassette consisting of chloramphenicol- and erythromycin-resistance genes was spliced into each construct to generate the pMEU shuttle vectors. Electrotransformation of Streptococcus thermophilus with these vectors showed that a ca. 1.7 kb BstEII/ BanII fragment is essential for plasmid replication. A shuttle vector, pMEU149-1 (5.3 kb), was constructed using the minimally required fragment for replication. A chloramphenicol acetyltransferase ( cat) gene was successfully expressed in the ultimate S. thermophilus host by using pMEU149-1. Cloning vectors derived from pER371 should provide valuable alternative gene delivery vehicles for the genetic engineering of lactic acid bacteria.

Metabolic engineering of sugar catabolism in lactic acid bacteria.

Lactic acid bacteria are characterized by a relatively simple sugar fermentation pathway that, by definition, results in the formation of lactic acid. The extensive knowledge of traditional pathways and the accumulating genetic information on these and novel ones, allows for the rerouting of metabolic processes in lactic acid bacteria by physiological approaches, genetic methods, or a combination of these two. This review will discuss past and present examples and future possibilities of metabolic engineering of lactic acid bacteria for the production of important compounds, including lactic and other acids, flavor compounds, and exopolysaccharides.

Heterologous expression and stability of the Escherichia coli β-galactosidase gene in Streptococcus lactis by translation fusion

An Escherichia coli/Streptococcus lactis shuttle vector for creating translation fusions to the E. coli galactosidase ( lacZ) has been constructed. Random S. lactis chromosomal DNA fragments inserted upstream of the lacZ gene and in frame promote the expression of active galactosidase in both E. coli and S. lactis. One fusion pSK216 has been characterized by Western blotting to reveal a fusion protein of 116 kd. S. lactis LM0230, a plasmidless lac − derivative of S. lactis C2 when carrying pSK216, exhibits a Lac + phenotype suggesting the presence of a chromosomally encoded lactose permease. Further evidence for this lactose permease system is provided by assay of lactose uptake by S. lactis LM0230. In the absence of selection pSK216 was less stable in S. lactis LM0230 than its derivative pSK41. The growth rate of S. lactis LM0230 carrying pSK216 is slower in comparison to the plasmid-free strain. Two genetic events were observed, the deletion of the lacZ gene and the loss of the entire plasmid. These results indicate that recombinant plasmids are unstable in S. lactis and imply that there is an obstacle in the genetic engineering of lactic acid bacteria.