Citation: Kazi, T.A.; Acharya, A.; Mukhopadhyay, B.C.; Mandal, S.; Arukha, A.P.; Nayak, S.; Biswas, S.R. Plasmid-Based Gene Expression Systems for Lactic Acid Bacteria: A Review. Microorganisms 2022, 10, 1132. https://doi.org/10.3390/ microorganisms10061132 Academic Editor: Alex Galanis Received: 28 April 2022 Accepted: 28 May 2022 Published: 31 May 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). microorganisms Review Plasmid-Based Gene Expression Systems for Lactic Acid Bacteria: A Review Tawsif Ahmed Kazi 1 , Aparupa Acharya 1 , Bidhan Chandra Mukhopadhyay 1 , Sukhendu Mandal 2 , Ananta Prasad Arukha 3 , Subhendu Nayak 4 and Swadesh Ranjan Biswas 1, * 1 Department of Botany, Visva-Bharati University, Santiniketan 731235, West Bengal, India; prince.evankazi@gmail.com (T.A.K.); aparupa.acharya@gmail.com (A.A.); bidhutntd@gmail.com (B.C.M.) 2 Laboratory of Molecular Bacteriology, Department of Microbiology, University of Calcutta, 35, Ballygunge Circular Road, Kolkata 700019, West Bengal, India; sukhendu1@hotmail.com 3 Researcher 5 Department of Neurosurgery, Medical School, University of Minnesota, Minneapolis, MN 55455, USA; arukh001@umn.edu 4 Sr. Scientist, Clorox, Better Health VMS, Durham, NC 27701, USA; subhendu.nayak@clorox.com * Correspondence: swadesh_s2000@yahoo.co.in; Tel.: +91-9064144574 Abstract: Lactic acid bacteria (LAB) play a very vital role in food production, preservation, and as probiotic agents. Some of these species can colonize and survive longer in the gastrointestinal tract (GIT), where their presence is crucially helpful to promote human health. LAB has also been used as a safe and efficient incubator to produce proteins of interest. With the advent of genetic engineering, recombinant LAB have been effectively employed as vectors for delivering therapeutic molecules to mucosal tissues of the oral, nasal, and vaginal tracks and for shuttling therapeutics for diabetes, cancer, viral infections, and several gastrointestinal infections. The most important tool needed to develop genetically engineered LABs to produce proteins of interest is a plasmid-based gene expression system. To date, a handful of constitutive and inducible vectors for LAB have been developed, but their limited availability, host specificity, instability, and low carrying capacity have narrowed their spectrum of applications. The current review discusses the plasmid-based vectors that have been developed so far for LAB; their functionality, potency, and constraints; and further highlights the need for a new, more stable, and effective gene expression platform for LAB. Keywords: lactic acid bacteria; plasmid-based vectors; NICE system; theta-mode of replication; rolling-circle replication; constitutive expression 1. Introduction Lactic acid bacteria represent a family of non-pathogenic, non-sporulating, microaerophilic, Gram-positive bacteria that produce lactic acid as the major end resultant during carbohy- drate fermentation and have been employed in food supplements, medicines, and cosmetics for ages to promote modern human civilization [1,2]. LAB are generally recognized as safe (GRAS) and due to this status, these food-grade Gram-positive bacteria have been used as starter cultures for food fermentation and as cell factories for the production of various macromolecules, enzymes, and relevant metabolites in the food, pharmaceutical, and dietary supplement industries [3–7]. These microbes not only support the fermentation process but also improve the organoleptic and rheological properties of food products [8]. Furthermore, due to their ability to produce various bacteriocins, they also help to extend the shelf life of various foods [4,9–12]. LAB are used as probiotics to promote human health as they maintain and modulate the intestinal flora; prevent hypersensitivity reactions and stimulate the immune response; and help protect against pathogens by producing antibacte- rial peptides, such as bacteriocin, in the gut [13]. Probiotic lactic acid bacteria are also used to treat several diseases such as diarrhea, inflammatory bowel diseases, and autoimmune diseases [14–17]. Because of their ability to efficiently produce recombinant proteins, LAB Microorganisms 2022, 10, 1132. https://doi.org/10.3390/microorganisms10061132 https://www.mdpi.com/journal/microorganisms