Tailoring cellular metabolism in lactic acid bacteria through metabolic engineering

Journal of Microbiological Methods 170 (2020) 105862

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Review

Tailoring cellular metabolism in lactic acid bacteria through metabolic engineering

T

Anshula Sharmaa, Gaganjot Guptaa, Tawseef Ahmada, Baljinder Kaura, , ⁎⁎ Khalid Rehman Hakeemb,c, ⁎

a

Department of Biotechnology, Punjabi University, Patiala 147002, India Department of Biological Sciences, Faculty of Science, King Abdulaziz University, 21589 Jeddah, Saudi Arabia c Princess Dr. Najla Bint Saud Al-Saud Center for Excellence Research in Biotechnology, King Abdulaziz University, Jeddah, Saudi Arabia b

ARTICLE INFO

ABSTRACT

Keywords: Central carbon metabolism Heterologous gene expression Synthetic metabolic engineering Systems biology Biosynthetic pathways Metabolite production

Metabolic engineering combines approaches of synthetic and systems biology for tailoring the existing and creating novel biosynthetic metabolic pathways in the desired industrial microorganisms for production of biofuels, bio-materials and environmental applications. Lactic acid bacteria (LAB) are gaining attention worldwide due to their extensive utilization in food, fermentation and pharmaceutical industries owing to their GRAS status. Well-elucidated genetics and regulatory control of central metabolism make them potential candidates for the production of industrially valuable metabolites. With the recent advancements in metabolic engineering strategies, genetic manipulation and tailoring of cellular metabolism is being successfully carried out in various LAB strains as they are providing highly efficient and industrially competitive robust expression systems. Thus, this review presents a concise overview of metabolic engineering strategies available for the comprehensive tailoring of lactic acid bacterial strains for large-scale production of industrially important metabolites.

1. Introduction 1.1. Biological activities and industrial relevance of lactic acid bacteria Lactic acid bacteria (LAB) constitutes an important Clostridium related clade of non-spore-forming, catalase-negative and microaerophilic Gram-positive species belonging to genera Aerococcus, Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Oenococcus, Pediococcus, Streptococcus, Tetragonococcus, Vagococcus and Weissella (Johnson-Green, 2002; Hutkins, 2006). Based on the mode of glucose fermentation, these are divided into two group i.e. homo-fermentative and hetero-fermentative organisms. Homo-fermentative lactic acid bacteria convert carbohydrates into lactic acid as a sole end product, while hetero-fermentative species also produce ethanol, acetic acid and carbon dioxide in addition to lactic acid (Halasz, 2009). Apart from the production of lactic acid, LAB also has the ability to produce industrially valuable metabolites having potential applications as nutraceuticals, pharmaceutics, commodity chemicals, aroma, flavour compounds and also in fermentation and waste processing industries as summarized in Fig. 1. Antimicrobial agents and metabolites such as ⁎

acetaldehyde, acetone, bacteriocins, benzaldehyde, diacetyl, formaldehyde, propanoic acid which contribute positively to aroma, flavour, stability, shelf life and texture of fermented foods (Lavermicocca et al., 2000; Leroy and De Vuyst, 2004; Routray and Mishra, 2011; Papagianni, 2012a). Since ancient times, lactic acid bacteria are used as starter cultures in fermented foods and introduced to the raw material as functional food ingredients for improving nutritive quality and safety of the end product besides conferring several health benefits being “Probiotic” in nature(Konings et al., 1999; Hansen, 2002; Shah, 2007). Besides food production, LAB are also reported to produce several industrially important enzymes viz. glycosylase, peptidases, amylases (Novik et al., 2007; Patel et al., 2012; Guldfeldt et al., 2001), vitamins like folate, B12, K2, riboflavin and thiamine (LeBlanc et al., 2011). Several lowcalorie sweeteners are vital food ingredients mainly marketed as “diabetic foods” produced using special or modified strains of LAB. For example, production of L-alanine, mannitol, sorbitol, xylitol, tagatose, and trehalose has been observed in LAB which can be incorporated directly to foods or can be generated in situ during LAB fermentation (Wisselink et al., 2002; Patra et al., 2009).

Corresponding author at: Baljinder Kaur, Department of Biotechnology, Punjabi University, Patiala 147002, India. Correspondence to: Khalid Rehman Hakeem, Department of Biological Sciences, Faculty of Science, King Abdulaziz University, 21589 Jeddah, Saudi Arabia. E-mail addresses: [email protected] (B. Kaur), [email protected] (K.R. Hakeem).

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https://doi.org/10.1016/j.mimet.2020.105862 Received 4 September 2019; Received in revised form 3 February 2020; Accepted 3 February 2020 Available online 05 February 2020 0167-7012/ © 2020 Elsevier B.V. All rights reserved.

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Fig. 1. Lactic acid bacteria as a source of industrially important compounds.

2. Metabolic engineering strategies for tailoring cellular metabolism in lactic acid bacteria

However, genetic engineering of LAB strains have mainly been aimed for food-based applications, but the potential use of recombinant LAB strains have been limited due to safety and stability aspects (Borner et al., 2019; Liu et al., 2019).To overcome this, more advanced and specific gene manipulation tools have been applied for the development of highly efficient and acceptable LAB strains to widen their industrial scope and utilization. Thus, efficient LAB cell factories are being developed by optimization and tailoring of desired metabolic pathways through systems metabolic engineering and synthetic biology approaches for the production of biofuels, biomaterials and food ingredients (Liu et al., 2019). Owing to their small genome size (~2–3 Mb) and simplicity in sugar and energy metabolism, LABs are considered as important candidates for metabolic engineering(Helando et al., 2005). During past two decades, considerable advancements have been made in the development of host-specific gene expression systems and efficient cloning protocols for the construction of highly robust expression systems for the production of industrially valuable metabolites(Papagianni, 2012a). Cellular metabolism in LAB has evolved in such a way that supports microbial growth and maintenance. Extensive robustness of cellular metabolism in LABis mainly attributed to the genetic redundancy and tightly regulated gene expression and metabolic processes. So, when metabolic engineering strategies are employed, the intrinsic regulatory networks of the cell strive to maintain homeostasis underplay to keep a check on its cellular metabolism even under altered intrinsic or extrinsic conditions. Therefore, genetic manipulations generally do not adversely affect cellular metabolism and biosynthesis of cellular components in LAB (Nielsen and Keasling, 2016). Metabolic engineering of LAB have focused on re-routing pyruvate metabolism and other complex biosynthetic pathways to produce commercially important end products like antimicrobial agents, aroma and flavour compounds, exopolysaccharides, sugar alcohols and sweeteners, nutraceuticals such as vitamins and other valuable metabolites with enormous health benefits (Kleerebezem and Hugenholtz, 2003; Sybesma et al., 2004; Nyyssola et al., 2005; Smid et al., 2005; Berlec and Strukelj, 2009; Mazzoli et al., 2014; Sauer et al., 2017; HattiKaul et al., 2018). Moreover, many LAB strains enjoy GRAS status (Generally recognized as safe by the FDA) and thus are most preferable candidates for food and pharmaceutical applications (Sybesma et al., 2006). In addition, many LAB strains were being proposed as potential vaccine delivery vehicles for generating an appropriate immune response in humans and farm animals (Suebwongsa et al., 2016). Thus, we envisage that the development and integration of novel genetic or metabolic engineering strategies will encourage the researchers to further explore the immense industrial and medical potential of LAB strains. The existing state of art approaches and tools for altering metabolic activities in LAB are discussed in the subsequent paragraphs.

Microorganisms are the oldest inhabitants and most versatile resources for the production of beverages, fermented foods and thousands of valuable compounds with novel activities and structures. From the last few decades, there is a dramatic increase in the exploitation of microorganisms for the biosynthesis of metabolites involved in environmental, industrial and therapeutic applications. Earlier strain improvement techniques were used to improve performance of the classical fermentation processes and to overcome challenges associated with complex metabolic regulation, reduced cellular growth, viability and product toxicity upon overexpression. With the advent of computational and synthetic biology tools in the late 1980s and early 1990s, there has been an important breakthrough in understanding the complexity of cellular biotransformation pathways, flux distribution over primary metabolic nodes, the flow of fluxes in alternative pathways and regulatory mechanism of central metabolism. These discoveries can be credited for inventing the field of metabolic engineering that came into existence in the 1990s which enabled molecular biologists to introduce specific genetic modifications in the microbial strains for redirecting metabolic fluxes towards the desired metabolite without compromising growth and basal cellular activities (Bailey, 1991; Lee and Papoutsakis, 1999; Kacser and Acerenza, 1993). Metabolic engineering not only modifies or redirects the microbial metabolic fluxes but also results in the improvement of the physiological performance of industrial microbes (Fu and Li, 2010). ‘Metabolic engineering’ deals with the direct improvement or modifications of the cellular processes for desired metabolite production in microorganisms (Nielsen and Keasling, 2011).Tremendous increase in fermentation productivity and the reduction in production costs of important cellular metabolites can be achieved by amalgamating classical genetic engineering techniques with the advanced metabolic engineering strategies as summarized in Fig. 2 (BarriosGonzalez et al., 1993; Adrio and Demain, 2006; Chae et al., 2017; Erb et al., 2017; Sharma et al., 2019a). Following developments in classical strain improvement techniques, highly precise metabolic deregulation can be ascertained in biological processes through gene duplication, protoplast fusion, targeted/random mutagenesis and classical gene recombination procedures such as conjugation, transposition and transposition, to meet the increasing demand of microbial metabolites at the industrial scale (Schwab, 1988; Adrio and Demain, 2010). More precise metabolic flux rerouting can be achieved by successful byproduct elimination, co-factor and promoter engineering, gene overexpression, gene knock-outs, heterologous expression of genes resistant to endproduct inhibition, precursor optimization and transporter engineering (Hugenholtz et al., 2000; Kleerebezem et al., 2000; Strohl, 2001; Nissen et al., 2016; Hoefnagel et al., 2002; Guo et al., 2012; Kulkarni, 2016). 2

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Fig. 2. Metabolic engineering strategies for tailoring cellular metabolism in lactic acid bacteria.

Advanced gene manipulation strategy like single-stranded DNA recombineering (SSDR) has also been successfully adopted in LAB strains to further explore their medical and industrial potential. Through this technology, if a rate-limiting step is identified in a pathway, then the same enzyme coding gene can be overexpressed by genetic engineering to accelerate the rate-limiting step in the concerned biotransformation pathway. Sometimes, the success rate in SSDR technique is limited due to intrinsic regulatory forces and genetic instability of the constructs within the cells and thus optimal expression may or may not be achieved in the recombinants (Pijkeren and Britton, 2014).In order to overcome these drawbacks, designing recombinants with integrated gene cassettes into chromosomal DNA has been recommended for industrially relevant LAB strains (Douglas and Klaenhammer, 2011). Highly efficient and advanced techniques viz. evolutionary engineering, synthetic metabolic engineering and systems biology have also been employed for improved and cost-effective production of desired microbial metabolites (Fig. 2)(Chae et al., 2017; Erb et al., 2017; Bar-Even et al., 2012; Sharma et al., 2019a). Evolutionary engineering methods are powerful tools for optimizing specificities of natural enzymes for their native and novel substrates and tailoring the already existing enzymes for the generation of completely new enzymes with novel and improved functional characteristics. Directed evolution, rational design, and semi-rational design approaches have been used widely in evolutionary engineering. Optimization of a computationally designed enzyme can be achieved by directed evolution through the screening of libraries of random mutations. Similarly, rational or semi-rational approaches can be applied for the creation of targeted libraries for the screening of metabolically active recombinant strains (Bar-Even et al., 2012). Adaptive evolution, metabolic or oligo mediated evolution and chimeric enzyme generation techniques have also been used for evolutionary engineering for tailoring and improving the catalytic attributes of enzymes for industrial

applications(Sharma et al., 2019a). Among synthetic metabolic engineering strategies, novel/non-natural pathway construction, pathway regulation and optimization, micro-compartment approach, co-culture technique, a spatial organization using DNA, RNA or protein scaffolds and CRISPR based gene modulations are the most preferred techniques (Erb et al., 2017). Systems biology is the most advanced and recent approach that interprets cellular phenomena at the molecular level with the help of more diversified and powerful tools such as omics data analysis and computational simulations. It employs comprehensive omics data analyses, identification of gene knockout targets, genomescale metabolic modelling, flux response analysis and in silico pathway prediction which are more systematic and high-throughput engineering methods (Chae et al., 2017). Classical metabolic engineering strategies such as “copy, paste, and fine-tuning” rely mainly on altering properties of a naturally existing metabolic pathway or extension of the partial biosynthetic pathway in the heterologous host by replacement or introduction of enzyme coding genes from other organisms for achieving desired metabolic activity. In contrast, synthetic metabolic engineering strategies are “mix and match” approaches based on the creation of non-native or synthetic metabolic pathways by recombining existing proteome with synthetic enzymes and native cellular metabolome. Synthetic or novel metabolic pathways constructed by the implementation of synthetic metabolic engineering strategies are more efficient than naturally existing ones in optimizing and redirecting cellular fluxes for the production of nonnatural desired biochemicals (Kumar and Prasad, 2011). Such strategies often lead to small alterations in the overall metabolic network and hence offer significant advantages such as improved substrate bioavailability, cellular viability, reaction kinetics, thermodynamic profile and cofactor utilization (Kumar and Prasad, 2011; Sharma et al., 2019a). So, the capabilities of native microorganisms can be extended and further explored for the establishment of more advanced and 3

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Fig. 3. Central carbon metabolism and Phosphotransferase (PT) systems for production of important metabolites in Lactic Acid Bacteria where 1: Hexokinase; 2: Phosphohexose isomerase; 3: Pyruvate-formate lyase; 4: Acetyl-Co-A hydrolase; 5: Acetoacetyl Co-A thiolase; 6: Aldehyde dehydrogenase; 7: Alcohol dehydrogenase; 8: Alanine dehydrogenase; 9: α-acetolactate synthase; 10: α-acetolactate decarboxylase; 11: Diacetyl reductase; 12: Acetoin reductase; 13: Mannitol-1-P dehydrogenase; 14: Sorbitol-6-P dehydrogenase; 15: Lactate dehydrogenase; 16: Malolactic enzyme; 17: Phosphoglucomutase: 18: GalU: UDP-glucose pyrophosphorylase; 19: GalE: UDP-galactose-4-epimerase; 20: Phosphoserine phosphatase; 21: Serine hydroxymethyltransferase; 22: β-glucosidases; 23: Malolactic bacteria; 24: Phosphotransacetylase; 25: Pyruvate Kinase.

efficient synthetic metabolic pathways by smartly implementing the “novel enzyme chemistries” with de novo enzyme designing (Chae et al., 2017; Erb et al., 2017; Sharma et al., 2019a). This has broadened the opportunities to create efficient cell factories especially LAB strains for the production of industrially valuable metabolites.

citric acid cycle as shown in Fig. 3. For harnessing biological properties of the cell at the industrial scale, it is imperative to have a good understanding of the intricacies and regulatory control of complex pathway reactions in CCM, the opportunity of novel product formation through genetic manipulations and significant flux rerouting towards the precursors for high-level metabolite production (Papagianni, 2012b). In a living cellular system, overall distribution of metabolic fluxes (especially the primary metabolites) is generally controlled by its genetic constitution and any disturbance caused due to genetic manipulation will provoke an inhibitory response within the cell which is either manifested in terms of reduced growth or hampered cellular metabolism, ultimately affecting the physiology of the cell as a whole (Sanchez and Demain, 2008; Klumpp and Hwa, 2014). Therefore, it is necessary to achieve the desired increase in flux while keeping metabolism and other important functions of the cell unchanged (Stephanopoulos and Sinskey, 1993). Another biggest experimental challenge for the implementation of metabolic engineering is the integration of genes encoding enzymes having diverse evolutionary and physiological histories for novel/non-

2.1. Challenges in implementing metabolic engineering strategies Despite the fact that metabolic engineering has a vast potential of improving the cellular traits of the desired microorganism, implementation of metabolic engineering strategies for development of high-performance cell factories that meet economical challenges for industrial-scale production is still experimentally challenging (Bailey, 1991; Stephanopoulos and Sinskey, 1993; Nielsen, 2001; Nielsen and Keasling, 2016). A direct concern of the metabolic engineering is to understand the nature, flux distribution, and regulatory mechanism of metabolic pathways associated with a microorganism. It requires extensive knowledge of the central carbon metabolism (CCM) which mainly involves the flow of carbon fluxes through primary metabolic pathways including glycolysis, gluconeogenesis, pentose pathways and 4

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homolactic to homoalanine fermentation. Similarly, in ldh− mutants of L. lactis, sugar metabolism was diverted using nisin inducible a-acetolactate decarboxylase towards the production of a-acetolactate which is a precursor of diacetyl. In this way, high diacetyl production from glucose was achieved(Hugenholtz et al., 2000).Moreover, complete disruption of ldh gene can lead to mannitol production in homofermentative LAB such as L. lactis and Lactobacillus plantarum as reported earlier (Ferrain et al., 1996; Neves et al., 2000).These studies provide convincing evidence in support of the fact that in case of homofermentative LAB, metabolism is mainly focused on lactic acid production but with the use of advanced metabolic engineering strategies, it is possible to redirect metabolic flux from lactate towards the production of other desirable metabolites.

natural pathway construction. Thus, sometimes this hampers the regulation and kinetics of the concerned pathway which ultimately limit the efficiency of the novel or synthetic pathways. Formation of inhibitory side-products or dead-end metabolites due to cross-inhibition while expressing the synthetic enzymes for specific pathway within a non-native host is also of great concern. But the potential solution for this problem is the integration of more specific isoenzymes, or homologs, so as to minimize the side reactions and undesirable side-product formation.The revolutionary enzyme engineering techniques can also be helpful in enhancing the enzyme specificity and efficiency for maximizing the occurrence of main or desirable reactions by tailoring of existing or by the construction of novel enzymes. Thus, engineering of native enzymes also provides great opportunities for the development of highly efficient, novel and stable enzyme catalysed pathways for desired industrial applications(Sharma et al., 2019b).Metabolic proofreading has also been helpful in the elimination of toxic side products, or recycling of the dead-end metabolites by the addition of specific proofreading enzymes to the core sequence of the concerned synthetic pathway. Micro-compartment approach can also be helpful in spatial separation of overlapping metabolic pathways and has an additional advantage of removal of toxic or reactive pathway intermediate thus, prevents the damage of cellular machinery. Thus, an amalgamation of conventional metabolic engineering techniques with more sophisticated synthetic metabolic engineering, systems biology and evolutionary engineering has greatly helped in overcoming the historical and ecological constraints of natural pathway evolution and has facilitated the production of desired and valuable metabolites in a great way(Sharma et al., 2019a).

4. Metabolic engineering of native LAB strains for industrially valuable metabolite production Metabolic engineering approaches in LAB have mainly focused on overexpression of the gene encoding rate-limiting enzyme of the concerned biosynthetic pathway, inhibition of the competing metabolic reactions, engineering of existing biosynthetic pathways or channelling of metabolic flux towards the biosynthesis of the desired chemical (Hugenholtz and Smid, 2002).Subsequent paragraphs highlight literature-based curation of the state of art metabolically engineering strategies for modulating metabolic activities of native LAB strains for production of industrially valuable metabolites (summarized in Table. 1). 4.1. Acetaldehyde

3. Homofermentative LAB: favoured strains for the production of desired metabolites

Acetaldehyde is an industrially valuable metabolite, widely used for imparting aroma and flavour in dairy products especially in yoghurt. In yoghurt bacterium Streptococcus thermophillus, overexpression of glyA gene encoding serine hydroxymethyltransferase (SHMT) enzyme having threonine aldolase (TA) activity, resulted in overproduction of acetaldehyde by 80 to 90% as compared to the parental strains. Threonine aldolase is reported to be responsible for the interconversion of threonine into acetaldehyde and glycine. In S. thermophilus, SHMT is the only enzyme reported to have TA activity and the main pathway for acetaldehyde formation is through the activity of SHMT. The results indicated that in the absence of an alternative pathway for acetaldehyde production, the overexpression of glyA enhanced the TA activity and also the acetaldehyde and folic acid production in recombinants (Chaves et al., 2002).

Homofermentative lactic acid bacteria have a quite simple metabolism. Undernutritious and sugar-rich environment, LABallows rapid sugar conversion mainly into lactic acid. Homofermentative lactate metabolism has a high metabolic rate which is achieved mainly due to impressive kinetic properties of the highly active lactate dehydrogenase (LDH) that mainly converts pyruvate to lactic acid (Hugenholtz and Kleerebezem, 1999).Under anaerobic conditions, lactic acid bacteria shows homolactic metabolism. But, under unusual conditions such as carbohydrate limitation, reduced sugar metabolism and aerobic conditions, pyruvate can be converted into alternative end-products such as acetic acid, acetoin, acetaldehyde, alanine, formic acid, 2,3-butanediol, diacetyl and mannitol. Such unusual conditions induce lower LDH activity which under limiting levels of the reducing cofactor NADH leads to a shift of homolactic metabolism towards the production of other metabolites as major end-products (Kleerebezem et al., 2000; Kleerebezem and Hugenholtz, 2003). This has led to the idea that inhibition and complete disruption of LDH activity along with the overexpression of dedicated genes, could lead to complete redirection of metabolic flux from lactic acid to the desired product. This strategy has been successfully employed in Lactococcus lactis, where efficient rerouting of pyruvate metabolism towards other end products such as diacetyl, mannitol and alanine has been carried out. For example, in ldh− mutants of Lactococcus lactis, anaerobic conditions induce synthesis of acetic acid, formic acid, acetoin and ethanol as end-products. But under aerobic conditions, mutant strain mainly produces homoacetoin during fermentation(Kleerebezem et al., 2000).The most successful example of complete metabolic rerouting in homofermentative LAB was reported by Hols et al., 1999. Cloning and heterologous overexpression of nisin inducible alaD gene from Bacillus sphaericus in ldh− mutants of L. lactis resulted in the production of alanine as a major end product of glucose metabolism. It was observed that more than 99.5% of glucose was converted into alanine. With the further deletion of alanine racemase (alr gene), complete stereo-specific production of L-alanine was achieved. In this way, the lactococcal metabolism was converted from

4.2. Acetoin Acetoin production using dairy industry waste was efficiently achieved in genetically engineered Lactococcus lactis by additional ATP consumption (AAC) strategy via plasmid-based expression of the F1ATPase. Authors reported the beneficial effect of AAC strategy on acetoin yield and productivity and this could be one possible way to increase the flux in the desired pathway. Acetoin yield was also increased by fine-tuning of the F1-ATPase with a concomitant positive effect on biomass yield. By the further introduction of lactose plasmid pLP712 in the recombinant strain, efficient acetoin production with a titer of 157 mM (14 g/L) was achieved (Liu et al., 2016a). Biocompatible chemistry (BC) involves the use of non-enzymatic chemical reactions compatible with living organisms that have significant contribution in the production of non-native metabolites in metabolically engineered microorganisms. It is gaining increasing attention because of potential applications in the synthesis of valuable compounds by linking metabolic pathways to achieve redox balance and rescued growth. By successful implementation of BC strategy, (3S)acetoin production was achieved in metabolically engineered L. lactis strain under chemically altered respiratory conditions where ferric iron 5

L. L. L. L. L. L. L.

L. L. L. S. L. L.

2,3-butanediol Diacetyl

Ethanol EPS

6

Succinic acid Tagatose Vanillin

1.3-Propanediol Riboflavin and Folate Sorbitol

Mannitol

3-hydroxypropionic acid L-Lactic acid

3-hydroxypropionaldehyde

Folate

Streptococcus thermophillus Lactococcus lactis L. lactis

Acetaldehyde Acetoin (3S)-acetoin

Overexpression of ldhL gene and disruption of pfl gene Disruption of ldhD gene Inactivation of mannitol transport system Inactivation of ldhD and ldhL genes Deletion of mtlA or mtlF genes Random mutagenesis Over expression of ribG, ribH, ribB and ribA genes Pathway optimization Directed mutagenesis and strain selection Integration of gutF gene and deletion of ldhL gene Inactivation of mtlD and ldh genes and overexpression of stlDH gene Co-expression of PC and PEPCK genes Disruption of lacC or lacD genes Media optimization by inducer supplementation P. acidilactici BD16 with cloned fcs and ech genes

Enterococcus faecalis

Lactobacillus paracasei 7BL L. lactis Lb. fermentum L. lactis Leuconostoc pseudomesenteroides L. lactis Lb. diolivorans L. lactis Lb. casei Lb. plantarum Lb. plantarum NCIMB 8826 L. lactis Pediococcus acidilactici BD16 Synthetic gene construct

Lactobacillus reuteri Lb. helveticus Lb. plantarum L. lactis Lb. johnsonii Lb. helveticus L. lactis L. lactis

Overexpression of pab and folB, folKE, folP, folQ, folC genes Combinatoral approach alongwith overexpression of glycerol dehydratase gene Metabolic flux re-routing Inactivation of ldhD gene by chromosomal integration Overexpression of the ldhL gene High copy number of las operon genes Inactivation of ldhD gene Inactivation of ldhD gene UV mutagenization Increased PFK activity

Gene knockout alongwith redirection of metabolic flux Overexpression of fbp gene Overexpression of galU using nisin inducible promoter Overexpression of pgm and galU genes Overexpression of gchI gene Overexpression of folKE and gchI gene

Overexpression of glyA gene Additional ATP consumption (AAC) Biocompatible chemistry alongwith inactivation of competing product pathway Biocompatible chemistry Inactivation of αldB geneandoverexpression of ilvBN genes Overexpression of nox genes Deletion of ldh and over expression of nox genes Genome-scale flux modelling Co-factor and promoter engineering Biocompatible chemistry

Metabolic engineering strategies

L. lactis Lactobacillus diolivorans

lactis lactis lactis thermophillus lactis strain NZ9000 lactis subsp. cremoris MG1363

lactis lactis lactis lactis lactis lactis lactis

Native LAB strain

Metabolite

Table 1 Metabolic engineering of native LAB strains for industrial metabolite production.

300 mM L-lactate from crude glycerol with a yield of > 99% within 48 h Enhanced production Enhanced production Mannitol production alongwith pyruvate 30% conversion of glucose to mannitol 10% enhanced production yield Overproduction of riboflavin 95% yield (92 g/L) Simultaneous overproduction of both the vitamins 0.043 mol sorbitol/mol glucose 0.65 mol sorbitol/mol glucose 22-fold higher production Transformed to a tagatose producing strain 1.26 g L − 1 crude vanillin 4 fold higher production

10.6 g/L production Production of pure L-Lactic acid No significant increase Low level production Production of pure L-Lactic acid 20% enhanced production yield Overproduction of L-lactate Enhanced lactic acid production

6.7 g/L titer Enhanced production Redirection of pyruvate flux towards diacetyl Enhanced production Increased diacetyl production 4 fold higher production (95 mM or 8.2 g/L) and high yield (87% of the theoretical maximum) 41 g/L and a yield of 70% of the theoretical maximum High level production No significant increase 2-fold increase in EPS synthesis 3-fold enhanced production 10-fold increase in extracellular and 3-fold in total production level High folate levels 35.9 g/L production

80–90% production yield 14 g/L titer 5.8 g/L titer

Effect on the biosynthesis

Kuo et al., 2015 Neves et al., 2000 Aarnikunnas et al., 2003 Gaspar et al., 2004 Helando et al., 2005 Burgess et al., 2004 Lindlbauer et al., 2017a Sybesma et al., 2004 Nissen et al., 2016 Ladero et al., 2007 Tsuji et al., 2013 Rooijen et al., 1991 Kaur and Chakraborty, 2013 Kaur et al., 2014

Dishisha et al., 2014 Bhowmik and Steele, 1994 Ferrain et al., 1994 Davidson et al., 1995 Lapierre et al., 1999 Kyla-Nikkila et al., 2000 Bai et al., 2004 Papagianni and Avramidis, 2011 Doi, 2015

Wegkamp et al., 2007 Lindlbauer et al., 2017a, 2017b

Liu et al., 2016d Looijesteijn et al., 1999 Boels et al., 2001 Levander et al., 2002 Hugenholtz et al., 2002 Sybesma et al., 2003

Liu et al., 2016c Benson et al., 1996 Hugenholtz et al., 2000 Hoefnagel et al., 2002 Oliveira et al., 2005 Guo et al., 2012 Liu et al., 2016c

Chaves et al., 2002 Liu et al., 2016a Liu et al., 2016b

Study

A. Sharma, et al.

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served as a BC component. Inactivation of competing for product pathways and appropriate cofactor balancing was carried out by finetuning of the respiratory capacity of the cells. The overall strategy eventually led to high-level (3S)-acetoin production with a final titer of 66 mM (5.8 g/L) (Liu et al., 2016b).

4.5. Ethanol L. lactis was metabolically engineered for the production of ethanol as the sole fermentation product from lactose substrate contained in residual whey permeates (RWP). Lactose catabolism was introduced into a L. lactis strain CS4435 (Δ3ldh, Δpta, ΔadhE, pCS4268) and the metabolic flux was redirected towards ethanol instead of lactate. Efficient ethanol production with a titer of 41 g/L and a yield of 70% of the theoretical maximum was obtained by employing fed-batch strategy using a low-cost medium formulation (Liu et al., 2016d).

4.3. Diacetyl Diacetyl which provides the typical butter aroma to the dairy products can also be generated from pyruvate. In the case of Lactococcus lactis, diacetyl production was enhanced by the inactivation of α-acetolactate decarboxylase gene (αldB) encoding ALDB enzyme. Further, the overexpression of the ilvBN genes (α-acetolactate synthase gene, ILVBN) in L. lactis led to enhanced levels of diacetyl. It was reported that aldB enzyme has a lower affinity for pyruvate which limits the diacetyl production. So, the inactivation of this gene was carried out to avoid enzymatic conversion of the diacetyl precursor α- acetolactate to acetoin followed by the overexpression of ilvBN genes which was reported to have a greater affinity for pyruvate (Benson et al., 1996). Sugar metabolism can be shifted from lactate production towards the production of α-acetolactate by either disruption of lactate dehydrogenase or overproduction of NADH oxidase as reported in a study. With the further disruption of the aldB gene, very effective diacetyl production from glucose and lactose under aerobic conditions led to the about 80% rerouting of the pyruvate pool towards α-acetolactate and diacetyl(Hugenholtz et al., 2000). Later on, Hoefnagel and co-workers adopted a rational combination strategy involving deletion of ldh and overexpression of nox genes which increased pyruvate flux from 0 to 75% of the measured diacetyl formation rates. The rational combination strategy consisted of three different approaches. One is the detailed kinetic modelling of the branches around pyruvate metabolism; the second one is the metabolic control analysis (MCA) and third is the experimental analysis or validation to describe the various mutations. These metabolic strategies were also aimed to successfully improve the suspected rate-limiting steps (Hoefnagel et al., 2002). In another study, Guo and co-workers manipulated pyruvate to lactate flux through co-factor and promoter engineering for the efficient production of lactate and diacetyl in L. lactis. NADH oxidase (NOX) activity increased to 58.17-fold by the use of selected promoters for the constitutive expression of nox gene under aerobic conditions by randomizing the promoter sequence of the H2Oforming NADH oxidase gene in L. lactis. For the constitutive expression of the H2O-forming nox gene in L. lactis, highly specific eleven promoters of the library were selected for promoter engineering which has led to an increase in the NADH oxidase activity in wild type strain from 9.43 to 58.17-fold under aerobic conditions. The authors reported that the reduced pyruvate to lactate flux was rerouted to the diacetyl with an almost linear flux variation via altered NADH/NAD+ ratios (Guo et al., 2012).

4.6. Exopolysaccharides Exopolysaccharides (EPSs) have been widely used in the production and improvement of fermented dairy products as they contribute to taste, texture, mouth-feel and stability of the dairy products (Arena et al., 2006; Kodali and Sen, 2008). The biosynthetic pathway for EPS production is divided into four separate reactions; sugar transport into the cytoplasm, the synthesis of sugar-1-phosphates, activation and coupling of sugars and secretion/export of the EPS. Genetic constitution and environment both contribute immensely to type and diversity of EPS production in LAB (Madhuri and Prabhakar, 2014). Sugar nucleotide diphosphates act as the precursors for EPS biosynthesis which are the main intermediates in central carbon metabolism (Boels et al., 2001). EPSs biosynthesis involves various genes coding for enzymes and main regulatory proteins as shown in Fig. 4. Further details on the biosynthesis of EPS in bacteria can be extracted from (Van Kranenburg et al., 1999; Jolly et al., 2002; Madhuri and Prabhakar, 2014; Cui et al., 2016). Exopolysaccharides production in LAB has also been enhanced by the use of various metabolic strategies. In the case of L. lactis, overexpression of the FBP(fructose bisphosphatase) gene led to enhanced levels of nucleotide sugars and EPSduring growth on fructose-containing media. FBP gene converts the fructose-1,6-diphosphate into fructose-6-phosphate, which is an essential step in the biosynthesis of sugar nucleotides from fructose but not from glucose which showed that the enzyme is highly substrate-specific (Looijesteijn et al., 1999).In another study, even eightfold increase in the levels of UDPglc and UDPgal by homologous overexpression of galU gene in L. lactis did not lead to enhanced EPS production. Authors thus reported that the EPS produced by this strain contains rhamnose sugar and the availability of dTDP-rhamnose precursor and eps gene cluster enzymes limit EPS biosynthesis in L. lactis (Boels et al., 2001).In S. thermophillus, overexpression of pgm (phosphoglucomutase) and galU (UDP-glucose phosphorylase) genes led to 2-fold increase in EPS synthesis by altering the expression of enzymes that supply major EPS precursors. A proportional increase in the EPS yield was observed when the levels of pgm and galU genes were increased together. But when either of the enzymes was expressed alone, no significant effect was seen on the EPS production (Levander et al., 2002). 4.7. Folate

4.4. Diacetyl and butanediol

Metabolic engineering has been employed to enhance the production levels of vitamin folate in L. lactis strain. Overexpression of gchI gene encoding GTP cyclohydrolase I, which is the first enzyme in folate biosynthesis in L. lactis strain NZ9000, led to a 3-fold increased production of folate as its overexpression increased the flux through the pathway and the enzyme, is not regulated by feedback inhibition. The ratios of monoglutamyl-folate over polyglutamyl-folate were also altered which further resulted in the higher release of folate into the environment (Hugenholtz et al., 2002). Overexpression of folKE that encodes the dimeric 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase and gchI genes in L. lactis subsp. cremoris MG1363, led to almost 10-fold increase in the extracellular level and 3-fold increase in the total production level of folate. Most of the folate produced by L.

Biocompatible chemistry in combination with metabolic engineering has significant contribution towards biosynthesis of valueadded biochemicals as well as in the extension of metabolic activities of the cells. Homolactic L. lactis strain was converted into homo-diacetyl producer by comprehensive rerouting of metabolism, activation of respiration and metal ion catalysis. Enhanced diacetyl production was obtained with a high titer (95 mM or 8.2 g/L) and yield (87% of the theoretical maximum). Subsequently, the pathway was extended towards (S, S)-2,3-butanediol (S-BDO) by linking their dedicated metabolic pathways via chemical catalysis which resulted in efficient homoS-BDO production with a titer of 74 mM (6.7 g/L) S-BDO(Liu et al., 2016c). 7

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Fig. 4. Metabolic pathway for Exopolysaccharides (EPS) production where; SPH: sucrose-6-phosphate hydrolase; FK: fructokinase; GalE: UDP-galactose-4-epimerase; GalK: galactokinase; GalTF: galactosyltransferase;GalU: UDP-glucose pyrophosphorylase; GlcTF: glucosyltransferase;PM: phosphomutase; RmlB: dTDP-glucose 4, 6dehydratase; RTF: rhamnosyltransferase; TGP: dTDP-glucose pyrophosphorylase; TRS: dTDP-4-dehydrorhamnose3,5-epimerase; UDP-GalNAc: UDP-N-acetylgalactosamine; UDP-GlcNAc: UDP-N-acetylglucosamine; UGT: UDP-glucuronosyltransferase; PLMM: phosphoglucosamine mutase; LacZ: β-glalactosidase; GTF: glycosyltransferase; GPN: glucosamine-1-phosphateN-acetyltransferase; GPD: glucosamine-6-phosphate deaminase; GLMU: N-acetylglucosamine-1-phosphate uridyltransferase; GlcTF: glucosyltransferase; UGDH: UDP-glucose 6-dehydrogenase; HXK: Hexokinase; LacS: lactose transporter; GalM: mutarotase; LDH: lactate dehydrogenase; PGI; phosphoglucoisomerase; PMI: phosphomannose isomerase; FTF: fucosyltransferase; PGM: α-phosphoglucomutase; GMD: GDP-mannose-4,6dehyratase; WcaG: GDP-4-keto-6-deoxy-mannose-3,5-epimerase-4-reductase.

lactisis intracellularly accumulated in the polyglutamyl form and this polyglutamylation is responsible for the retention of folate within the cell. The enzymatic capacity of folate synthetase/polyglutamyl folate synthetase was not enough enhanced by the overexpression of folKE to transform all the extra produced folate into polyglutamyl form which has led to decreased folate retention (Sybesma et al., 2003). Higher intracellular folate levels can only be achieved through simultaneous overexpression of the folate biosynthesis gene cluster including folB, folKE, folP, folQ, folC genes and the pab genes for para-aminobenzoic acid (pABA) biosynthesis. It was observed that elevated pABA pools inhibit the activity of the folylpolyglutamate synthetase enzyme which is responsible for increased polyglutamate tails and higher intracellular retention (Wegkamp et al., 2007).

4.8. 1,3-propanediol, 3-hydroxypropionaldehyde and 3-hydroxypropionic acid 1,3-PDO is an industrially important metabolite, widely been used in adhesives, cosmetics, detergents, resins and solvents (Zeng and Sabra, 2011). In several heterofermentative LAB such as Lb. brevis, Lb. buchneri, Lb. reuteri and Lb. diolivorans, glycerol acts as an electron acceptor and gets itself converted to 1,3-propanediol by regeneration of NAD(P)H along with enhanced growth rate and biomass yield (Pflügl et al., 2012). 1,3-PDO production from glycerol through propanediolutilization pathway has been reported in various LAB such as Lactobacillus species, Streptococcus sanguinis and Enterococcus malodoratus (Chen and Hatti-Kaul, 2017). Efficient production of 1,3-PDO in Lb. diolivorans was achieved by pathway optimization strategy of metabolic engineering.Also, by co-feeding glucose or lignocellulose hydrolysates, 92 g/L of high titer was achieved with 95% yield. However, the expensive downstream processing, complex recovery processes and 8

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formation of other side products such as organic acids and ethanol, limits the large scale production of 1,3-PDO (Lindlbauer et al., 2017a). 3-hydroxypropionaldehyde(3-HPA) and 3-hydroxypropionic acid (3-HP) are important platform chemicals having potential applications in bio-based industries (Bozell and Petersen, 2010). From the genome sequencing of Lactobacillus reuteri, it was revealed that glycerol dehydratase (PduCDE) enzyme is mainly involved in the first step of glycerol metabolism in which glycerol gets converted to 3-HPA by dehydration which is further reduced to 1,3-PDO by the activity of 1,3-PDO oxidoreductase (PduQ) enzyme (Sriramulu et al., 2008). Enhanced production of 3-HPAfrom crude glycerol in Lactobacillus diolivorans was achieved by employing combinatorial approach of metabolic engineering. By overexpression of endogenous glycerol uptake facilitating gene encoding glycerol dehydratase enzyme, 35.9 g/L of 3-HPA production was obtained in the recombinant LAB strain (Lindlbauer et al., 2017b). Lb. reuteri has been reported to be a potential candidate for 3HP production because of its high acid tolerance and ability to produce coenzyme B12naturally which are the major essentials for 3-HP production. 3-HP production in Lb. reuteri revealed the existence of a parallel oxidative branch of the Pdu pathway for 3-HPA metabolism. The enzymes mainly involved in the pathway for 3-HP production are coenzyme-A acylating propionaldehyde dehydrogenase (PduP), phosphotransacylase (PduL) and propionate kinase (PduW) (Sabet-Azad et al., 2013; Dishisha et al., 2014). Lb. reuteri strain was engineered by mutating the catabolite repression element (CRE) to improve the redirection of the metabolic flux of glycerol towards 3HP and 1,3-propanediol production. Metabolic flux analysis revealed that glycerol dehydration to 3-HPA was ten times faster than the subsequent oxidation and reduction of 3-HPA to 1,3-propanediol and 3HP. Thus, by optimizing the flux rate of glycerol towards 3-HPA and re-directing the glycerol flux towards 3HP and 1,3-propanediol,3-HPA accumulation decreases. By using resting cells of Lb. reuteri under anaerobic and fedbatch conditions, 10.6 g/L 3HP and 9.0 g/L 1,3-propanediol production were achieved (Dishisha et al., 2014).

metabolite was linked to an increase in glucose uptake rate and reduction of NADH oxidase activity (Bai et al., 2004). During lactic acid fermentation, there is a possibility for the synthesis of either racemic mixture of D- or L-lactic acid or one of them which is being controlled by isomer-specific enzymes namely D- and L-lactate dehydrogenases. The racemase activity was observed in certain LAB strains (e.g. L. plantarum and L. sakei) which were found to be responsible for the conversion of L-lactate into D-lactate (Goffin et al., 2005).In the case of Lactococcus lactis, the direct effect of PFK (phosphofructokinase) activity on the glycolytic flux was observed. Specific rates of glucose uptake and lactate formation were increased with a twofold increase in PFK activity (Papagianni and Avramidis, 2011). Metabolically engineered Enterococcus faecalis strain W11 was explored for efficient conversion of crude glycerol to L-lactate. Authors reported that engineered strain could use biodiesel waste as a carbon source, although cell growth was significantly inhibited by the oil component in the biodiesel waste, which decreased the cellular NADH/ NAD+ ratio and then induced oxidative stress to cells. The lactate dehydrogenase (ldh) activity of W11 strain was 4.1-fold lower under anaerobic glycerol metabolism than that during aerobic glycerol metabolism which eventually led to low L-lactate productivity. The E. faecalis mutant strain (the ∆pfl mutant) having the glycerol-inducible ldhL1LP gene expression plasmid, produced 300 mM L-lactate from glycerol/crude glycerol with a yield of > 99% within 48 h and reached maximum productivity of 18 mM h1(Doi, 2015). Lactic acid production from lignocellulosic feedstock is quite difficult as substrate pretreatment process releases many toxic substances which inhibit microbial growth and activities. However, lactic acid production from lignocellulosic biomass was successfully achieved in Lactobacillus paracasei 7BL which showed high tolerance to inhibitors and optically pure L-lactic acid was produced after the interruption of ldhD gene. The strain 7BL fermented glucose efficiently and showed high titer of L-lactic acid (215 g/L) by fed-batch strategy. In addition, 99 g/L of l-lactic acid with high yield (0.96 g/g) and productivity (2.25–3.23 g/L/h) was obtained by using non-detoxified wood hydrolysates (Kuo et al., 2015).

4.9. Lactic acid

4.10. Mannitol

Production of pure L-lactic acid in homofermentative or heterofermentative LAB has been successfully carried out by various strategies of metabolic engineering. Production of pure L-lactic acid in Lactobacillus helveticus was achieved after inactivation of the D-lactate dehydrogenase encodingldhD gene by chromosomal integration (Bhowmik and Steele, 1994).In L. lactis, Davidson and co-workers reported a small increase in L-lactic acid productionby introducing additional copies of las operon genes including the ldhL, phosphofructokinase (pfk) and pyruvate kinase (pyk) genes (Davidson et al., 1995). Similarly, in Lb. plantarum, overexpression of L-LDH gene had hardly any effect on the production of L-(+)- and D-(−)-lactate. Further inactivation of gene encoding L-LDH enzyme led to the production of only D isomer of lactate. However, the global concentration of lactate in this bacterium remained unchanged. On the other hand, inactivation of ldhD gene resulted in suppression of both D and L-lactate production in Lb. plantarum mutant strain(Ferrain et al., 1994; Ferrain et al., 1996). Lactate production in pure L- form was reported in Lb. johnsonii by the inactivation of ldhD gene but some pyruvate flux was also diverted to other end products such as diacetyl and acetoin (Lapierre et al., 1999). In L. helveticus, inactivation of ldhD gene resulted in 20% enhanced production of L-(1)-lactic acid under low pH conditions. Two stable ldhD-negative L. helveticus derivates were constructed using gene replacement method. Internal deletion of the promoter region has led to the prevention of transcription of ldhD gene. Further, lactic acid production and growth were controlled by pH cultivation (Kyla-Nikkila et al., 2000). In a separate study, L-lactate production was enhanced by UV mutagenesis of wild type L. lactis strain. However, overproduction of the

Mannitol is an important sugar alcohol with a multitude of applications in various industrial bioprocesses such as artificial sweetener,flavour modifier, anti-caking and texturizing agent. In pharmaceutical industries, mannitol is being widely used to mask bitterness of various antibiotics and used as an antioxidant, anti-cancerous and osmotic diuretic agent(Hugenholtz et al., 2002; Ortiz et al., 2017). It has been reported thatmannitol biosynthesis is practically nonexistent in L. lactis under normal conditionsprobably due to catabolic repression. Neves and co-workers provided convincing evidences to prove that inactivation of mannitol transport system in ldh-deficient L. lactis strain led to enhanced mannitol production and mannitol-1-phosphate (Mtl1P). After depletion of glucose, mannitol is quickly metabolized and diauxic growth pattern is followed by the cells. Mannitol-1-phosphate dehydrogenase reaction isused as NAD+is no longer regenerated in the reaction where pyruvate is reduced to lactate (Neves et al., 2000). Further, deletion of mtlA or mtlF genes or disruption of parts of the phosphoenolpyruvate (PEP)-mannitol phosphotransferase system (PTSMtl) by double-crossover recombination in ldh-deficient L. lactis resulted in almost 30% glucose conversion to mannitol in resting cells of the double mutant. Even after complete glucose depletion, the double mutants were unable to utilize mannitol as mannitol transport in L. lactiswas blocked by the gene disruption. To further enhance mannitol production in L. lactis, pathway engineering was carried out to reduce diversion of metabolic flux to ethanol, 2,3-butanediol, and lactate biosynthesis(Gaspar et al., 2004). Simultaneous cloning and stepwise inactivation of ldhD and ldhL genes using gene replacement system in Lb. fermentum strain have led to 9

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production of mannitol in combination with pyruvate or pure L-lactic acid. In single mutant GRL1030, mannitol and lactic acid yields were unaffected by deletion of ldhD gene. In double mutant GRL1032, mutations negatively affected glucose consumption rate and resulted in reduced cellular growth.Double mutant strain produced 2,3-butanediol and some lactic acid in addition to production of mannitol and pyruvate as expected(Aarnikunnas et al., 2003). In Leuconostoc pseudomesenteroides, a combinatorial approach involving inactivation of fructokinase activity and random mutagenesis resulted in enhanced production of mannitol from fructose from 74 to 85% (mol/mol). This is because of the fact that decreased activity of fructokinase can effectively reduce the leakage of fructose into the phosphoketolase (PK) pathway (Helando et al., 2005).

introducing modifications in their biosynthetic pathways following strategies of directed mutagenesis and strain selection. The pathways were diverted from pyruvate towards desired vitamin biosynthesis (Sybesma et al., 2004). 4.14. Vanillin Metabolic rerouting for induction of specific metabolite production in native microbial cells can also be achievedby provision of specific enzyme inducers. This strategy was employed for strain improvement in a lactic acid bacterial isolate Pediococcus acidilactici BD16 for the production of vanillin. Vanillin production byP. acidilactici BD16 was induced in an enrichment medium containing 150 g/L rice bran and 50 μg/Lferulic acid.Both ferulate containing substrate i.e. rice bran and inducer of vanillin biosynthetic operon i.e. ferulic acid were utilizedfor the production of 1.26 g/L crude vanillin in the native bacterial cells (Kaur and Chakraborty, 2013).Vanillin biosynthesis was further enhanced by cloning a synthetic vanillin gene cassette, bearing fcs and ech genes encoding feruloyl-CoA synthetase and enoyl-CoA hydratase in P. acidilactici BD16. After metabolic engineering and scale up of the process, 4 g/L vanillin was recovered from recombinant cell culture whichwas almost four fold higher than the native strain (Kaur et al., 2014).Also, recombinant P. acidilactici BD16 (fcs+/ech+) was further explored to enhance aroma and flavour characteristics of wine by malolactic fermentation due to production of several phenolic metabolites including vanillin (Kaur et al., 2015). In a parallel study, vanillin biosynthetic gene cassette was also introduced in E. coli Top 10 (T7fcs+/ech+) using genetic manipulation techniques. Crude enzyme extracts of the recombinant E. coli Top 10 (T7-fcs+/ech+) were used in a lab scale ferulic acid biotransformation studies and 68 mg/L vanillin per 10 mg/L ferulic acid was obtained after 30 min of biotransformation, with no subsequent vanillin degradation (Chakraborty et al., 2016).

4.11. Sorbitol In Lb. casei, integration of sorbitol-6-phosphate-dehydrogenase gene (gutF) into the chromosomal lactose (lac) operon resulted in the production of 0.024 mol sorbitol/mol glucose as compared to production of negligible amounts in the parental strain. The regulation of gutF gene expression was observed to be same as that of lac genes, where glucose inhibits its production through catabolite repression, while lactose acts as an inducer. Due to which when cells were pre-grown on lactosecontaining medium; sorbitol was produced from glucose. With further deletion of ldhL gene, sorbitol production was increased to 0.043 mol per mol glucose and the authors suggested that this engineering strategy provides an alternative pathway for NAD+ regeneration (Nissen et al., 2016).Sorbitol production in Lb. plantarum and Lb. casei strains was also achieved through an improved metabolic engineering strategy.For example, inactivation of mtlD (mannitol phosphate dehydrogenase) and ldh genes and overexpression of stlDH (sorbitol dehydrogenase gene, SDH) resulted in enhanced bioconversion of fructose-6phosphate to sorbitol-6-phosphate by SDH and theoretical yield of 0.65 mol sorbitol/mol glucosewas attained inLb. plantarum strain.With further enhancement of dephosphorylation and transport (export) of sorbitol, even higher levels of sorbitol production were achieved (Ladero et al., 2007).

5. Metabolite production in non-native LAB strains through heterologous gene expression Despite employing all the possible genetic manipulative procedures and metabolic engineering strategiesin native strains,sometimes the production titers of the desired metaboliteare still low or not up to the demand.In such cases, the best feasible solution is the transfer ofconcernedmetabolic pathway to new/non-native hosts, that is, a heterologous host for enhanced production of desired or non-natural metabolites. Thus, multiple genes encoding specific enzymes for the concerned pathway can be expressed in a non-native host for novel pathway construction or rewiring of central metabolic fluxes for optimization of existing pathways for high-level production of the native or desired metabolite in the non-native host strains(Sharma et al., 2019a).Heterologous gene expression has been successfully carried out in non-native LABstrains for improved production of cellular metabolites (Table. 2).

4.12. Succinic acid Lactobacillus plantarum NCIMB 8826 strain has been reported to possess a defective in tricarboxylic acid cycle (TCA) and consequently produce small amounts of succinic acid naturally. The lactate dehydrogenase-deficient strain of L. plantarum NCIMB 8826 (VL103) was metabolically engineered for enhanced succinic acid production. Authors observed the affect of overexpression or coexpression of potential enzymes viz. pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK), and malic enzyme on succinic acid production. Results supported the fact that biosynthesis of succinic acid was mainly due to the activity of PC enzymewhereasPEPCK enzyme majorly led to increased biomass in L. plantarum NCIMB 8826 (VL103). Ultimately, coexpression of PC and PEPCK led to highest succinic acid yield in L. plantarumVL103. 22-fold higher succinic acid production was achieved in recombinant strain as compared to the wild-type strainwith 25.3% conversion of glucose to succinic acid (Tsuji et al., 2013).

5.1. Acetaldehyde Enhanced production of acetaldehyde by heterologous expression was reported by Bongers and co-workers in L. lactis. Overexpression of Zymomonas mobilis pyruvate decarboxylase (pdc) gene in L. lactis led to the rerouting of the pyruvate metabolism towards acetaldehyde. And further overexpression of NADH oxidase (nox) gene under anaerobic conditions, 50% conversion rate of glucose was reported in the resultant double mutant strain (Bongers et al., 2005).

4.13. Riboflavin In case of L. lactis subsp. cremoris strain NZ9000, high level riboflavin synthesis was carried out by simultaneous over expression of four riboflavin biosynthetic genes namely ribG, ribH, ribB and ribA using gene-specific genetic engineering strategy. Targeted metabolic engineering and isolation of spontaneous mutants to a toxic riboflavin analogue were used to achieve the overproducing riboflavin L. lactis strain (Burgess et al., 2004).Simultaneous overproduction of folate and riboflavin in L. lactis was also reported by Sybesma and co-workers by

5.2. Alanine Heterologous production of alanine in L. lactis NZ9000 was carried out by expression of alanine dehydrogenase (ALDH) gene (alaD) from 10

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Liu et al., 2017 Solem et al., 2013 Gilbert et al., 1998 Van Kranenburg et al., 1999 Li et al., 2015 Germond et al., 2001 Werning et al., 2008 Stingele et al., 1999 Papagianni and Avramidis, 2011 Wisselink et al., 2005 361 mM R-BDO with 81% yield Ethanol as major end product Enhanced production Enhanced EPS synthesis 219.4 mg/L; enhanced (46%) production EPS production starts 300 mg/L synthesis of (133)-β-D-glucan Enhanced EPS production 22.8 g lactate (g CDW)−1 h−1; enhanced (2 fold) production* Enhanced 67% production L. lactis with butanediol dehydrogenase gene L. lactis MG1363 expressing pdc gene L. lactis expressing cpu gene cluster L. lactis NIZO B40 with epsD gene Lactobacillus casei LC2W with cloned nox gene L. lactis MG1363with eps gene cluster L. lactis with overproduction of gtf gene L. lactis MG 1363 with Sfi6 eps gene cluster L. lactis with cloned pfkA gene (two fold increase in PFK activity)

Eom et al., 2010 Srikanth and Halami, 2008 Humbelin et al., 1999 Nyyssola et al., 2005

Ye et al., 2010 Kandasamy et al., 2016 L. lactis NZ9000 pNZ273/ldhp/ald L. lactis with lactose plasmid pLP712

B. subtilis Enterobacter cloacae, Achromobacter xylosooxidans. Bacillus subtilis Z. mobilis Streptococcus pneumoniae S. pneumonie S. mutans S. thermophilus Pediococcus parvulus S. thermophilus Aspergillus niger

Pediocin PA-1 synthesis starts Improved production 3 fold enhanced production 2.5 mol xylitol/mol glucose

Bongers et al., 2005 Hols et al., 1999

50% conversion of glucose to acetaldehyde 12.6 g/L L-alanine was produced from 1.8% w/ v glucose 52 μg/mL; enhanced (26 fold) production 51 and 32 g/L production Lactococcus lactis with overexpression of pdc and nox genes L. lactis NZ3950withexpression of alaD anddisruption of ldhA gene Zymomonas mobilis Bacillus sphaericus IFO3525

5.3. Acetoin and butanediol L. lactis has proven to be an excellent cell factory for transforming lactose-containing dairy waste into industrially important metabolites. Biosynthetic metabolism of L. lactis was manipulated and diverted towards the production of (3R)-acetoin, meso-(2,3)-butanediol (m-BDO) and (2R,3R)-butanediol (R-BDO). Efficient production of (3R)-acetoin was accomplished by blocking the competing pathways for lactate, acetate and ethanol production. Higher production levels of m-BDO or R-BDO were achieved by heterologous expression of different alcohol dehydrogenases, either EcBDH from Enterobacter cloacae or SadB from Achromobacter xylosooxidans. Further transformation of the above strains with the lactose plasmid pLP712 enabled efficient production of (3R)-acetoin, m-BDO and R-BDO from processed whey waste, with titers of 27, 51, and 32 g/L respectively (Kandasamy et al., 2016). In another study, acetoin and R-BDO production were optimized in metabolically engineered L. lactis by fine-tuning of a respiratory mechanism involving a switch from fully respiratory to non-respiratory conditions during dual-phase fermentation. Fully activated respiration efficiently regenerated NAD+ which led to enhanced production of acetoin with titer of 32 g/L. Consequently, the metabolic pathway from acetoin to R-BDO was extended by heterologous expression of the butanediol dehydrogenase gene from Bacillus subtilis and 361 mM R-BDO with a yield of 81% or 365 mM with a yield of 82% was obtained respectively (Liu et al., 2017). 5.4. Ethanol Ethanol production was successfully achieved in L. lactis MG1363 by introduction of codon-optimized Zymomonas mobilis pyruvate decarboxylase (pdc) gene expressed using synthetic promoters. It was observed that recombinantL. lactisproduced lower amounts of ethanol when grown on glucose even after introduction of the pdc gene, probably due to its low native alcohol dehydrogenase activity. Further, coinactivation of the lactate dehydrogenase genes namely ldhX, ldhB, and ldh resulted in synthesis of ethanolas the major product along with small amounts of lactate, formate, and acetate when double engineered strainwas grown on maltose containing medium. Subsequent inactivation oftwo genes coding for phosphotransacetylase and a native alcohol dehydrogenasefinally led to production of ethanol as the sole fermentation product (Solem et al., 2013).

Bifidobacterium adolescentis P. acidilactici K7 Bacillus subtilis Pichia stipitis and Lb. brevis

Lb. plantarum and Eimeria tenella

L. lactis with over expression of mtlD gene and mannitol 1-phosphate phosphatase gene Lactobacillus reuteri KCTC 3679 L. lactis MG1363 expressing ped A and pedB immunity genes L. lactis with overexpressionof ribA gene L. lactis with co-expression of xylI gene and xylose transporter

Reference Outcomes of the study Heterologous host with modifications Native host

Bacillus subtilis. Results showed that over-expression of heterologous alaD gene in L. lactis NZ9000 carrying pNZ273/ldhp/ald enhanced alanine levels to 52 mg/mL which was 26-fold higher compared to the parent strain (Ye et al., 2010). Interestingly, the Km for pyruvate in B. subtilis ALDH was reported as 0.44 mM which is much lower than the Kmreported for pyruvate in L. lactis L-LDH (1.15 mM).This suggests that ALDH of B. subtilis is more competitive than L-LDH of L. lactis for the substrate pyruvate. Therefore, the introduction of more copies of the gene encoding B. subtilis ALDH into L. lactis host strain and its higher Km, both led to better and enhanced alanine production in the recombinant strain as compared to the recombinant strain reported by Hols et al., 1999.

In a study, the heterologous production of bacteriocin pediocin PA-1 in L. lactis MG1363 was carried out by expressing pedA structural and pedB immunity genes from the native Pediococcus acidilactici K7strain and reported to have enhanced bacteriocin production (Srikanth and Halami, 2008). In another study, Pediocin PA-1 structural and immunity genes (pedAB) fused inframe with the promoter and deduced signal sequence of an α-amylase gene from a bifidobacterial strain, cloned in Lactobacillus reuteri KCTC 3679 also resulted in improved bacteriocin activity in recombinant strain (Eom et al., 2010).

Riboflavin Xylitol

Pediocin PA-1

Mannitol

Lactic Acid

EPS

2,3-butanediol and 2R,3Rbutanediol (R,R)-2,3-butanediol Ethanol EPS

Acetaldehyde Alanine

5.5. Pediocin PA-1

Metabolite

Table 2 Heterologous production of industrially important metabolites in non-native LAB strains.

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5.6. Exopolysaccharides

competitive LAB strains. Cellular metabolism in LAB has been rigorously studied and manipulated by researchers since long, for the development of industrially viable, robust and competitive expression systems with desired and improved traits. LAB are excellent hosts for the expression of heterologous attributes which facilitated their exploration as microbial cell factories for the production of industrially important metabolites. Under natural conditions, the stoichiometry of the complex cellular pathways in LAB is controlled by its genome as well as environmental pressures, thus they may not be able to show the optimized responses in order to grade them as industrial competitive for product synthesis. Under such circumstances, existing biosynthetic pathways can be manipulated or novel pathways can be constructed by heterologous gene expression or following advanced genetic manipulation procedures to manipulate well-controlled cellular metabolism and to target the desired metabolic flux nodes for overproduction of existing or novel metabolites. Tailoring cellular metabolism in LAB through metabolic engineering approach has, in many instances, resulted in the development of bioprocesses with enhanced catalytic efficiencies, productivities and specificities. However, in future, to further widen its scope and applications, multiplex and robust genome manipulation tools are needed for costeffective and large scale production of valuable metabolites in LAB. Genome-scale metabolic modelling has already been successfully implimentedfor the re-construction of efficient metabolic networks and flux optimization. Also,combinatorial approaches focussing synthetic systems engineering and genome-scale metabolic remodelling can assist in creation of novel products and enzymes with improved catalytic attributes and higher expression levels. In addition, more accurate and reliable protein structure modelling and kinetics prediction tools can be introduced for assisting metabolic engineering and enzyme remodelling. Thus, it is anticipated that in future that advanced next-generation techniques and efficient metabolic remodelling strategies will be developed and practised for better understanding and tailoring of cellular metabolism in LAB for novel, exciting and valuable applications.

Heterologous expression of the complete S. thermophilus Sfi6 eps gene cluster has been achieved in a non-exopolysachharide (EPS)-producing strain of L. lactis MG 1363. This strategy yielded an EPS with a different composition and structure from the EPS produced in native host strain(Stingele et al., 1999). Heterologous expression of genes responsible for type 3 capsular polysaccharide (CPUs) production in S. pneumoniae has resulted in high-level production of type 3 polysaccharide in L. lactis (Gilbert et al., 1998). Homologous insertion of an epsD (gene coding for a priming glycosyl-transferase) of S. pneumonie into the mutated L. lactis NIZO B40 also resulted in enhanced EPS production (Van Kranenburg et al., 1999). Overexpression of the glycosyltransferase (GTF) gene of Pediococcus parvulus in an uncapsulated L. lactis strain resulted in the synthesis of 300 mg/L position 2-substituted (133)-β-D-glucan which is a potential food additive (Werning et al., 2008).In another study, cloning of eps gene cluster from S. thermophilus Sfi39 into a non-EPS-producing heterologous host L. lactis MG1363 resulted in EPS production as identical to the EPS produced by the native host (Germond et al., 2001). Improved EPS production by cofactor engineering was carried out in Lactobacillus casei LC2W, which is a potential EPS-producing strain with probiotic effects. Under the control of constitutive promoter P23, nox gene of Streptococcus mutanswas overexpressed in L. casei LC2W. The recombinant strain showed 20-fold enhanced NADH activity with 219.4 mg/L of EPS production which was increased by 46% compared to that of the wild-type strain (Li et al., 2015). 5.7. Lactic acid Heterologous expression in LAB has also led to enhanced lactic acid production levels. Papagianni and Avramidis observed the effects of increased PFK activity on the production of L-lactic acid by cloning the pfkA gene of Aspergillus niger into L. lactis strain. They reported an increase in specific PFK activity by 2 fold that increased from 7.1 to 14.5 U/OD600. There was also a significant increase in specific rates of glucose uptake which increased from 0.8 to 1.7 μMs−1 g CDW−1 and lactate formation which increased from 15 to 22.8 g lactate (g CDW)−1 h−1(Papagianni and Avramidis, 2011). Heterologous production of mannitol in the ldh-deficient L. lactis was carried out by the overexpression of mannitol 1-phosphate dehydrogenase gene (mtlD) of Lb. plantarum and the mannitol 1-phosphate phosphatase gene (M1Pase) of the protozoan parasite Eimeria tenella. The 50% conversion of glucose into mannitol was observed which is the highest reported conversion efficiency from glucose to mannitol with a theoretical yield of 67% in L. lactis(Wisselink et al., 2005). Overexpression of Bacillus subtilis riboflavin biosynthetic ribA gene coding for GTP cyclohydrolase II enzyme under NICE expression system has led to 3-fold overproduction of riboflavin in L. lactis (Humbelin et al., 1999).

Declaration of Competing Interest Authors declare no conflict of interest. Acknowledgement Authors acknowledge Indian Council of Medical Research, New Delhi, India for providing senior research fellowship to Ms. Anshula Sharma, vide no. 2017-3616/CMB/BMS. References Aarnikunnas, J., von Weymarn, N., Ronnholm, K., Leisola, M., Palva, A., 2003. Metabolic engineering of Lactobacillus fermentum for production of mannitol and pure L-lactic acid or pyruvate. Biotechnol. Bioeng. 82, 653–663. Adrio, J.L., Demain, A.L., 2006. Genetic improvement of processes yielding microbial products. FEMS Microbiol. Rev. 30, 187–214. Adrio, J.L., Demain, A.L., 2010. Recombinant organisms for production of industrial products. Bioeng. Bugs. 1 (2), 116–131. Arena, A., Maugeri, T.L., Pavone, B., Iannello, D., Gugliandolo, C., Bisignano, G., 2006. Antiviral and immunoregulatory effect of a novel exopolysaccharide from a marine thermotolerant Bacillus licheniformis. Int. Immunopharmacol. 6, 8–13. Bai, D.M., Zhao, X.M., Li, X.G., Xu, S.M., 2004. Strain improvement and metabolic flux analysis in the wild-type and a mutant Lactobacillus lactis strain for L(+)-lactic acid production. Biotechnol. Bioeng. 88, 681–689. Bailey, J.E., 1991. Toward a science of metabolic engineering. Sci. 252 (5013), 1668–1675. Bar-Even, A., Flamholz, A., Noor, E., Milo, R., 2012. Rethinking glycolysis: on the biochemical logic of metabolic pathways. Nat. Chem. Biol. 8, 509–517. Barrios-Gonzalez, J., Montenegro, E., Martin, J.F., 1993. Penicillin production by mutants resistant to phenylacetic acid. J. Ferment. Bioeng. 7, 455–458. Benson, K.H., Godon, J.J., Renauld, P., Griffin, H.G., Gason, M.G., 1996. Effect of ilvBNencoded α-acetolactate synthase expression on diacetyl production in Lactococcus lactis. Appl. Microbiol. Biotechnol. 45, 107–111. Berlec, A., Strukelj, B., 2009. Novel applications of recombinant lactic acid bacteria in therapy and in metabolic engineering. Recent Pat. Biotechnol. 3 (2), 77–87.

5.8. Xylitol Strains of S. avium and Lb. casei have been reported to metabolize xylitol but still cannot produce it naturally (London, 1990). Heterologous production of xylitol in L. lactis was carried out by Nyyssola and coworkers by the co-expression of xylose reductase (xylI) gene from Pichia stipitis and a xylose transporter from Lb. brevis. However, the strategy followed could not significantly improve xylitol production in the recombinant strain (Nyyssola et al., 2005). 6. Conclusion and future perspectives The interdisciplinary field of metabolic engineering is rapidly evolving with the advent of advanced systems biology, synthetic biology, and evolutionary engineering approaches, which has concurrently led to the development of high-performance and industrially 12

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