TY - JOUR
AU - Mazzoli, Roberto
AB - Abstract Lactic acid bacteria (LAB) have a long history in industrial processes as food starters and biocontrol agents, and also as producers of high-value compounds. Lactic acid, their main product, is among the most requested chemicals because of its multiple applications, including the synthesis of biodegradable plastic polymers. Moreover, LAB are attractive candidates for the production of ethanol, polyhydroalkanoates, sweeteners and exopolysaccharides. LAB generally have complex nutritional requirements. Furthermore, they cannot directly ferment inexpensive feedstocks such as lignocellulose. This significantly increases the cost of LAB fermentation and hinders its application in the production of high volumes of low-cost chemicals. Different strategies have been explored to extend LAB fermentation to lignocellulosic biomass. Fermentation of lignocellulose hydrolysates by LAB has been frequently reported and is the most mature technology. However, current economic constraints of this strategy have driven research for alternative approaches. Co-cultivation of LAB with native cellulolytic microorganisms may reduce the high cost of exogenous cellulase supplementation. Special attention is given in this review to the construction of recombinant cellulolytic LAB by metabolic engineering, which may generate strains able to directly ferment plant biomass. The state of the art of these strategies is illustrated along with perspectives of their applications to industrial second generation biorefinery processes. lactobacillus, lactococcus, cellulase, recombinant cellulolytic strategy, metabolic engineering, cellulosome INTRODUCTION Lactic acid bacteria (LAB) have extensive industrial applications, mainly in food fermentation and as probiotics (Mazzoli et al.2014). Relevant industrial processes involving LAB also include fermentative production of lactic acid (LA). LA is among the most requested chemicals because of its different applications in food (e.g. acidifier and flavour-enhancing agent), cosmetics (emulsifying and moisturising agent) and pharmaceutical (intermediate) industries, and as a building block for the synthesis of biodegradable plastic polymers (e.g. polylactides (PLAs)) (Abdel-Rahman, Tashiro and Sonomoto 2013). It has been estimated that about 90% of the worldwide LA is produced through LAB fermentation (Sauer et al.2008). LA can also be produced by chemical synthesis, but this gives rise to a racemic mixture of D- and L-LA which is not suitable for PLA production (Abdel-Rahman and Sonomoto 2016). Furthermore, D-LA can cause metabolic problems in humans and therefore cannot be used in the food, drink, and pharmaceutical industries (Jem, van der Pol and de Vos 2010). Depending on the specific LAB strain genome, i.e. the presence of gene(s) encoding D- or L-lactate dehydrogenase and/or racemase, D- or L-LA or their mixtures can be produced. In addition, LAB have been considered as candidates for production of other high-value compounds such as ethanol, polyhydroalkanoates, polyols and exopolysaccharides (Mazzoli et al.2014). However, most LAB are auxotrophic for several amino acids, nucleotides and vitamins (that should supplement their growth media). Furthermore, LAB, with a few exceptions, cannot ferment abundant inexpensive biomass such as starchy or lignocellulosic feedstocks. These are significant limits for LAB being applied to economically viable biorefinery processes, especially those aimed at producing high volumes of low-cost molecules (e.g. ethanol). Nowadays, most LA is produced by the bioconversion of dedicated crops (mainly corn) by companies such as Corbion-Purac (The Netherlands), Galactic (Belgium) and NatureWorks LLC-Cargill (USA) (Abdel-Rahman, Tashiro and Sonomoto 2013; de Oliveira et al.2018). As the global demand for LA is rapidly increasing (16.2% annual growth) (de Oliveira et al.2018), such a process represents a threat to these food crops. Development of fermentation processes based on second generation (i.e. lignocellulosic) feedstocks appears to be a priority for the extensive application of LAB in biorefinery. So far, no native cellulolytic and/or hemicellulolytic LAB has been identified. However, a number of LAB strains have been isolated from plant environments, e.g. from fermented vegetables or the gastrointestinal tract of herbivores where plant biomass is the main carbon source. These LAB developed the ability to ferment a variety of soluble sugars derived from plant polysaccharide hydrolysis (see next section). Supplementation of cellulases in the growth medium (Adsul, Varmab and Gokhale 2007; Wee and Ryu 2009; Shi, Kang and Lee 2015; Bai et al.2016; Hu et al.2016; Overbeck, Steele and Broadbent 2016; Wang et al.2017; Grewal and Khare 2018) or co-cultivation with cellulolytic microorganisms (Shahab et al.2018) have therefore been used as efficient strategies to allow plant biomass fermentation by LAB. Alternatively, the development of recombinant LAB equipped with heterologous cellulase systems has been pursued in order to obtain strains that can directly ferment lignocellulosic feedstocks (i.e. consolidated bioprocessing (CBP)) (Mazzoli et al.2014). The state of the art of these strategies and future research directions towards their application in industrial processes will be described in the following sections. THE ABILITY OF LAB TO FERMENT SOLUBLE MONO-/OLIGO-SACCHARIDES FROM LIGNOCELLULOSIC BIOMASS LAB can metabolise several monosaccharides, including both hexoses (e.g. fructose, glucose and galactose) and pentoses (e.g. xylose) (Kandler 1983), which are common components of lignocellulosic materials. Based on their metabolism, LAB are classified as homo-, hetero- and mixed acid-fermenters (Kandler 1983). In homofermentative metabolism, sugars are catabolised through the Embden-Meyerhof-Parnas pathway and converted to pyruvate which is finally reduced to LA. Heterofermentative metabolism involves sugar conversion through the phosphoketolase pathway giving rise to equimolar mixtures of LA and ethanol/or acetic acid (Kandler 1983). Finally, in mixed acid fermenters, glycolysis-derived pyruvate is metabolised through multiple pathways resulting in the production of LA and ethanol and/or acetic and/or formic acid mixtures (Kandler 1983). Efficient metabolism of pentose sugars is particularly important when hemicellulose fermentation is addressed (Jordan et al.2012). Some LAB such as Lactobacillus pentosus, Lactobacillus brevis, Lactobacillus plantarum and Leuconostoc lactis can metabolise both arabinose and xylose through heterofermentative metabolism (Fig. 1) (Tanaka et al.2002; Okano et al.2009a). An additional xylose fermentation pathway featuring higher LA production yields was identified in Lactococcus lactis IO-1 (Tanaka et al.2002). In this strain, at high xylose concentrations, xylose catabolism is shifted from the phosphoketolase pathway to the pentose-phosphate pathway, which catalyses its homo-lactic conversion (Fig. 1) (Tanaka et al.2002). Figure 1. View largeDownload slide Heterolactic (red) and homolactic (blue) pathways for xylose dissimilation in LAB. DHAP, dihydroxyacetone phosphate; Fba, fructose bisphosphate aldolase; GAP, glyceraldehyde-3-phosphate; Ldh, lactate dehydrogenase; Pkt, phosphoketolase; Pfk, 6-phosphofructokinase; Tal, transaldolase; Tkt, transketolase; Tpi, triose phosphate isomerase; XylA, xylose isomerase; XylB xylulokinase. Figure 1. View largeDownload slide Heterolactic (red) and homolactic (blue) pathways for xylose dissimilation in LAB. DHAP, dihydroxyacetone phosphate; Fba, fructose bisphosphate aldolase; GAP, glyceraldehyde-3-phosphate; Ldh, lactate dehydrogenase; Pkt, phosphoketolase; Pfk, 6-phosphofructokinase; Tal, transaldolase; Tkt, transketolase; Tpi, triose phosphate isomerase; XylA, xylose isomerase; XylB xylulokinase. Efficient metabolism of oligosaccharides derived from partial hydrolysis of cellulose/hemicellulose is essential for optimal fermentation of these polysaccharides (Galazka et al.2010; Lane et al.2015). In native cellulolytic microorganisms, a significant part of these oligosaccharides are most likely not saccharified in the extracellular environment (Desvaux 2006). Instead, they are transported through specific proteins into the cytoplasm where they are further metabolised by either the hydrolytic or phosphorolytic mechanism (Desvaux 2006). Notoriously, cellodextrin transport and intracellular metabolism have been engineered in important candidates for second generation biorefinery such as Saccharomyces cerevisiae and Yarrowia lipolytica (Galazka et al.2010; Lane et al.2015). Advantageously, an increasing number of natural LAB have been shown to metabolise cellobiose and other short cellodextrins or short oligosaccharides derived from hemicellulose (e.g. xylan, β-glucan) hydrolysis (Ohara, Owaki and Sonomoto 2006; Adsul, Varmab and Gokhale 2007; Kowalczyk et al.2008; Okano et al.2010b; Lawley, Sims and Tannock 2013). Recently, Lc. lactis IL1403, i.e. one of the most referenced LAB strains, has shown the natural ability to ferment up to cellotetraose/cellopentaose (Gandini et al.2017). This study has indicated that this strain is equipped with membrane transporters for short cellodextrins, although they have not been identified yet. The genome of this strain is rich in genes encoding putative β-glucosidases/6-P-β-glucosidases, while no gene coding for cellodextrin phosphorylase is present (Bolotin et al.2001). As regards the metabolism of partial hydrolysis products of hemicellulose, it is worth reminding the identification of three LAB strains, i.e. Lc. lactis IO-1, Leu. lactis SHO-47 and Leu. lactis SHO-54, that can ferment xylooligosaccharides with degrees of polymerisation up to six (Ohara, Owaki and Sonomoto 2006). Here again it was demonstrated that these xylooligosaccharides are hydrolysed by intracellular xylosidases, while transporters for their uptake were not identified (Ohara, Owaki and Sonomoto 2006). Although rare, the presence of genes encoding enzymes involved in the depolymerisation of xylooligosaccharides and/or arabinoxylans and/or arabinans (i.e. β-xylosidases and arabinofuranosidases) has been detected in different strains of Lactobacillus spp., Pediococcus spp., Leuconostoc/Weissella branch, and Enterococcus spp. (Michlmayr et al.2013). Recently, Lb. ruminis, an inhabitant of human bowels and bovine rumens, has been shown to ferment tetrasaccharides derived from barley β-glucan (Lawley, Sims and Tannock 2013). Since fermented vegetables and other environments rich in plant biomass are habitats in which LAB can be commonly found, it is likely that future analyses will identify further LAB strains equipped with basic biochemical systems for metabolising sugars derived from plant material. ALTERNATIVE STRATEGIES FOR LIGNOCELLULOSE FERMENTATION THROUGH LAB Fermentation of pre-treated lignocellulosic biomass by natural LAB Since natural LAB cannot directly hydrolyse and ferment polysaccharides present in lignocellulose, physical and/or chemical and/or enzymatic pre-treatment(s) of biomass are necessary. Several examples of fermentation of different pre-treated/hydrolysed lignocellulosic feedstocks by LAB have been reported that include de-oiled algal biomass (Overbeck, Steele and Broadbent 2016), barley bran (Moldes et al.2006), corncob (Guo et al.2010; Bai et al.2016), corn stover (Hu et al.2016; Wang et al.2017), de-oiled cottonseed cake (Grewal and Khare 2018), oak wood chip (Wee and Ryu 2009), paper mill sludge (Marques et al.2008; Shi, Kang and Lee 2015), sugarcane bagasse (Adsul, Varmab and Gokhale 2007; Laopaiboon et al.2010), trimming vine shoots (Bustos et al.2005; Moldes et al.2006), wheat bran (Naveena et al.2005; Li et al.2010) and wheat straw (Grewal and Khare 2018) (Table 1). Table 1. Examples of LAB fermentation of pre-treated lignocellulosic biomass. Strategies for physico-chemical and/or enzymatic pretreatment of biomass are summarised. n.r., not reported; SHF, separate hydrolysis and fermentation; SSF, simultaneous saccharification and fermentation. The term SSF has been employed for processes featuring simultaneous saccharification and fermentation of all the soluble sugars derived from biomass hydrolysis that, depending on the biomass composition, may be hexoses or pentoses or mixtures (i.e. co-fermentation). Biomass Physico-chemical treatment(s) Enzymatic treatment Microorganisms Fermentation mode LA enantiomer LA (g/L) Yield Y P/S (g/g) Productivity (g/L/h) Reference Algal cake (de-oiled algal biomass) - Porcine pepsin (37°C, 3 h) plus Lb. casei 12A SHF Batch L- (and traces of D-) 11.17 - - Overbeck, Steele and Broadbent 2016 α-amylase (37°C, 16 h) plus endo-1,4-β-D-glucanase (50°C, 24 h) from Aspergillus niger Barley bran Biomass was dried, milled and hydrolyzed with 3% H2SO4 (130°C, 15 min) - Lb. pentosus CECT-4023T SHF Batch n.r. 33 0.57a 0.60 Moldes et al.2006 Birch wood xylan - Xylanase (1.25 g/L) (60°C, 20 min) Leu. lactis SHO-47 SHF Batch D- 2.3 - - Ohara, Owaki and Sonomoto 2006 Corncob Biomass was dried, milled and hydrolysed with 2% H2SO4 (130°C, 15 min) - Lb. pentosus CECT-4023T SHF Batch n.r. 26 0.53a 0.34 Moldes et al.2006 Corncob Biomass was mashed and hydrolysed with 0.1% H2SO4 (80°C, 1 h) and 0.8% H2SO4 (110°C, 2 h) - Lb. brevis S3F4 SHF Batch n.r. 39.1 0.69a 0.81 Guo et al.2010 Corncob residue - Commercial cellulase mixture (15 FPU/g biomass) Sporolactobacillus inulinus YBS1–5 SHF Fed-Batch D- 107.2 0.85b 1.19 Bai et al.2016 Corn stover Biomass was mashed and hydrolysed with 2% H2SO4 at 10% (w/v) (121°C, 1 h) - Lb. brevis S3F4 SHF Batch n.r. 18.2 0.74a 0.76 Guo et al.2010 Corn stover Biomass was dried, sieved and treated with 5% NaOH (75°C, 3 h) Commercial cellulase, β-glucosidase, and xylanase mixture (30 FPU/g biomass) Lb. pentosus FL0421 SSF Fed-batch n.r. 92.30 0.66c 1.92 Hu et al.2016 Corn stover Biomass was crushed, sieved, dried and treated with 1.5% solid acid (120°C, 80 min) Commercial cellulase mixture (30 FPU/g biomass) Lactobacillus delbrueckii delbrueckii sp. bulgaricus CICC21101 SSF Batch D- 18 - Wang et al.2017 De-oiled cottonseed cake Biomass was ground, sieved and mixed with ionic liquid (120°C, 2 h) Immobilised cellulases (25 FPU/g biomass) from Trichoderma reesei Lactobacillus brevis MTCC 4460 SSF Batch n.r. - 0.22c - Grewal and Khare, 2018 Detoxified Eucalyptus globulus Biomass was dried, milled, and hydrolyzed with 3% H2SO4 (130°C, 1 h) - Lb. pentosus CECT-4023T SHF Batch n.r. 14.5 0.70a 0.28 Moldes et al.2006 Hydrolysate was neutralised with CaCO3 and stirred with 15% w/v of charcoal (room temperature, 1 day) Oak wood chip Biomass was treated with 0.5% H2SO4 (room temperature, overnight) and steam explosion (215°C, 5 h) Commercial cellulase mixture (20 IU/g) supplemented with β-glucosidase (30 IU/g) (50°C, 48 h) Lactobacillus sp. RKY2 SHF Continuous cell recycle (dilution rate 0.16 h−1) n.r. 42 0.95b 6.7 Wee and Ryu, 2009 Recycled paper sludge Biomass was neutralised with 0.3 g HCl/g biomass - Lb. rhamnosus ATCC 7469 SSF Batch n.r. 73 0.97a 2.9 Marques et al.2008 Softwood pre-hydrolysate plus paper mill sludge Softwood particles were sieved and pre-treated with hot water Commercial cellulases (15 FPU/g glucan) plus pectinases (15 mg protein/g mannan) Lactobacillus rhamnosus ATCC-10863 SSF Batch n.r. 60 0.83d 0.62 Shi, Kang and Lee 2015 Sugarcane bagasse Biomass shreds (1–3 mm) were pre-treated with steam and alkali Enzyme preparation from Penicillium janthinellum Lb. delbrueckii mutant Uc-3 SSF Batch L- 67 0.83e 0.93 Adsul, Varmab and Gokhale 2007 Sugarcane bagasse Biomass was dried, milled, treated with 10% NH4OH and hydrolysed with 0.5% HCl (100°C, 5 h). Hydrolysate was detoxified by amberlite treatment - Lc. lactis IO-1 JCM 7638 SHF Batch n.r. 10.9 - 0.14 Laopaiboon et al.2010 Sugarcane bagasse Biomass was ground, sieved and mixed with ionic liquid (120°C, 2 h) Immobilised cellulases (25 FPU/g biomass) from Trichoderma reesei Lactobacillus brevis MTCC 4460 SSF Batch n.r. - 0.52c - Grewal and Khare, 2018 Trimming vine shoots Biomass was dried, milled and hydrolysed with 3% H2SO4 (130°C, 15 min) - Lb. pentosus CECT-4023T SHF Batch n.r. 46 0.78a 0.933 Bustos et al.2005 Trimming vine shoots Substrate was dried, milled and hydrolysed with 3% H2SO4 (130°C, 15 min) - Lb. pentosus CECT-4023T SHF Batch n.r. 24 0.76a 0.51 Moldes et al.2006 Wheat bran Biomass was pre-reduced and sterilised - Lb. amylophilus GV6 Solid state fermentation L- - 0.23c - Naveena et al.2005b Wheat bran Biomass was treated with 1.5% H2SO4 (ratio 1:4 w/v) (80°C, 20 h) - Lb. rhamnosus LA-04–1 SHF Batch L- 75 0.99b 3.75 Li et al.2010b Wheat straw Biomass was ground, sieved and mixed with ionic liquid (120°C, 2 h) Immobilized cellulases (25 FPU/g biomass) from Trichoderma reesei Lactobacillus brevis MTCC 4460 SSF Batch n.r. - 0.49c - Grewal and Khare, 2018 Biomass Physico-chemical treatment(s) Enzymatic treatment Microorganisms Fermentation mode LA enantiomer LA (g/L) Yield Y P/S (g/g) Productivity (g/L/h) Reference Algal cake (de-oiled algal biomass) - Porcine pepsin (37°C, 3 h) plus Lb. casei 12A SHF Batch L- (and traces of D-) 11.17 - - Overbeck, Steele and Broadbent 2016 α-amylase (37°C, 16 h) plus endo-1,4-β-D-glucanase (50°C, 24 h) from Aspergillus niger Barley bran Biomass was dried, milled and hydrolyzed with 3% H2SO4 (130°C, 15 min) - Lb. pentosus CECT-4023T SHF Batch n.r. 33 0.57a 0.60 Moldes et al.2006 Birch wood xylan - Xylanase (1.25 g/L) (60°C, 20 min) Leu. lactis SHO-47 SHF Batch D- 2.3 - - Ohara, Owaki and Sonomoto 2006 Corncob Biomass was dried, milled and hydrolysed with 2% H2SO4 (130°C, 15 min) - Lb. pentosus CECT-4023T SHF Batch n.r. 26 0.53a 0.34 Moldes et al.2006 Corncob Biomass was mashed and hydrolysed with 0.1% H2SO4 (80°C, 1 h) and 0.8% H2SO4 (110°C, 2 h) - Lb. brevis S3F4 SHF Batch n.r. 39.1 0.69a 0.81 Guo et al.2010 Corncob residue - Commercial cellulase mixture (15 FPU/g biomass) Sporolactobacillus inulinus YBS1–5 SHF Fed-Batch D- 107.2 0.85b 1.19 Bai et al.2016 Corn stover Biomass was mashed and hydrolysed with 2% H2SO4 at 10% (w/v) (121°C, 1 h) - Lb. brevis S3F4 SHF Batch n.r. 18.2 0.74a 0.76 Guo et al.2010 Corn stover Biomass was dried, sieved and treated with 5% NaOH (75°C, 3 h) Commercial cellulase, β-glucosidase, and xylanase mixture (30 FPU/g biomass) Lb. pentosus FL0421 SSF Fed-batch n.r. 92.30 0.66c 1.92 Hu et al.2016 Corn stover Biomass was crushed, sieved, dried and treated with 1.5% solid acid (120°C, 80 min) Commercial cellulase mixture (30 FPU/g biomass) Lactobacillus delbrueckii delbrueckii sp. bulgaricus CICC21101 SSF Batch D- 18 - Wang et al.2017 De-oiled cottonseed cake Biomass was ground, sieved and mixed with ionic liquid (120°C, 2 h) Immobilised cellulases (25 FPU/g biomass) from Trichoderma reesei Lactobacillus brevis MTCC 4460 SSF Batch n.r. - 0.22c - Grewal and Khare, 2018 Detoxified Eucalyptus globulus Biomass was dried, milled, and hydrolyzed with 3% H2SO4 (130°C, 1 h) - Lb. pentosus CECT-4023T SHF Batch n.r. 14.5 0.70a 0.28 Moldes et al.2006 Hydrolysate was neutralised with CaCO3 and stirred with 15% w/v of charcoal (room temperature, 1 day) Oak wood chip Biomass was treated with 0.5% H2SO4 (room temperature, overnight) and steam explosion (215°C, 5 h) Commercial cellulase mixture (20 IU/g) supplemented with β-glucosidase (30 IU/g) (50°C, 48 h) Lactobacillus sp. RKY2 SHF Continuous cell recycle (dilution rate 0.16 h−1) n.r. 42 0.95b 6.7 Wee and Ryu, 2009 Recycled paper sludge Biomass was neutralised with 0.3 g HCl/g biomass - Lb. rhamnosus ATCC 7469 SSF Batch n.r. 73 0.97a 2.9 Marques et al.2008 Softwood pre-hydrolysate plus paper mill sludge Softwood particles were sieved and pre-treated with hot water Commercial cellulases (15 FPU/g glucan) plus pectinases (15 mg protein/g mannan) Lactobacillus rhamnosus ATCC-10863 SSF Batch n.r. 60 0.83d 0.62 Shi, Kang and Lee 2015 Sugarcane bagasse Biomass shreds (1–3 mm) were pre-treated with steam and alkali Enzyme preparation from Penicillium janthinellum Lb. delbrueckii mutant Uc-3 SSF Batch L- 67 0.83e 0.93 Adsul, Varmab and Gokhale 2007 Sugarcane bagasse Biomass was dried, milled, treated with 10% NH4OH and hydrolysed with 0.5% HCl (100°C, 5 h). Hydrolysate was detoxified by amberlite treatment - Lc. lactis IO-1 JCM 7638 SHF Batch n.r. 10.9 - 0.14 Laopaiboon et al.2010 Sugarcane bagasse Biomass was ground, sieved and mixed with ionic liquid (120°C, 2 h) Immobilised cellulases (25 FPU/g biomass) from Trichoderma reesei Lactobacillus brevis MTCC 4460 SSF Batch n.r. - 0.52c - Grewal and Khare, 2018 Trimming vine shoots Biomass was dried, milled and hydrolysed with 3% H2SO4 (130°C, 15 min) - Lb. pentosus CECT-4023T SHF Batch n.r. 46 0.78a 0.933 Bustos et al.2005 Trimming vine shoots Substrate was dried, milled and hydrolysed with 3% H2SO4 (130°C, 15 min) - Lb. pentosus CECT-4023T SHF Batch n.r. 24 0.76a 0.51 Moldes et al.2006 Wheat bran Biomass was pre-reduced and sterilised - Lb. amylophilus GV6 Solid state fermentation L- - 0.23c - Naveena et al.2005b Wheat bran Biomass was treated with 1.5% H2SO4 (ratio 1:4 w/v) (80°C, 20 h) - Lb. rhamnosus LA-04–1 SHF Batch L- 75 0.99b 3.75 Li et al.2010b Wheat straw Biomass was ground, sieved and mixed with ionic liquid (120°C, 2 h) Immobilized cellulases (25 FPU/g biomass) from Trichoderma reesei Lactobacillus brevis MTCC 4460 SSF Batch n.r. - 0.49c - Grewal and Khare, 2018 ag of LA/g of total sugar in the hydrolysate. bg of LA/g of glucose in the hydrolysate. cg of LA/g of biomass. dg of LA/g of total hexose sugars. eg of LA/g of cellulose in the biomass. View Large Table 1. Examples of LAB fermentation of pre-treated lignocellulosic biomass. Strategies for physico-chemical and/or enzymatic pretreatment of biomass are summarised. n.r., not reported; SHF, separate hydrolysis and fermentation; SSF, simultaneous saccharification and fermentation. The term SSF has been employed for processes featuring simultaneous saccharification and fermentation of all the soluble sugars derived from biomass hydrolysis that, depending on the biomass composition, may be hexoses or pentoses or mixtures (i.e. co-fermentation). Biomass Physico-chemical treatment(s) Enzymatic treatment Microorganisms Fermentation mode LA enantiomer LA (g/L) Yield Y P/S (g/g) Productivity (g/L/h) Reference Algal cake (de-oiled algal biomass) - Porcine pepsin (37°C, 3 h) plus Lb. casei 12A SHF Batch L- (and traces of D-) 11.17 - - Overbeck, Steele and Broadbent 2016 α-amylase (37°C, 16 h) plus endo-1,4-β-D-glucanase (50°C, 24 h) from Aspergillus niger Barley bran Biomass was dried, milled and hydrolyzed with 3% H2SO4 (130°C, 15 min) - Lb. pentosus CECT-4023T SHF Batch n.r. 33 0.57a 0.60 Moldes et al.2006 Birch wood xylan - Xylanase (1.25 g/L) (60°C, 20 min) Leu. lactis SHO-47 SHF Batch D- 2.3 - - Ohara, Owaki and Sonomoto 2006 Corncob Biomass was dried, milled and hydrolysed with 2% H2SO4 (130°C, 15 min) - Lb. pentosus CECT-4023T SHF Batch n.r. 26 0.53a 0.34 Moldes et al.2006 Corncob Biomass was mashed and hydrolysed with 0.1% H2SO4 (80°C, 1 h) and 0.8% H2SO4 (110°C, 2 h) - Lb. brevis S3F4 SHF Batch n.r. 39.1 0.69a 0.81 Guo et al.2010 Corncob residue - Commercial cellulase mixture (15 FPU/g biomass) Sporolactobacillus inulinus YBS1–5 SHF Fed-Batch D- 107.2 0.85b 1.19 Bai et al.2016 Corn stover Biomass was mashed and hydrolysed with 2% H2SO4 at 10% (w/v) (121°C, 1 h) - Lb. brevis S3F4 SHF Batch n.r. 18.2 0.74a 0.76 Guo et al.2010 Corn stover Biomass was dried, sieved and treated with 5% NaOH (75°C, 3 h) Commercial cellulase, β-glucosidase, and xylanase mixture (30 FPU/g biomass) Lb. pentosus FL0421 SSF Fed-batch n.r. 92.30 0.66c 1.92 Hu et al.2016 Corn stover Biomass was crushed, sieved, dried and treated with 1.5% solid acid (120°C, 80 min) Commercial cellulase mixture (30 FPU/g biomass) Lactobacillus delbrueckii delbrueckii sp. bulgaricus CICC21101 SSF Batch D- 18 - Wang et al.2017 De-oiled cottonseed cake Biomass was ground, sieved and mixed with ionic liquid (120°C, 2 h) Immobilised cellulases (25 FPU/g biomass) from Trichoderma reesei Lactobacillus brevis MTCC 4460 SSF Batch n.r. - 0.22c - Grewal and Khare, 2018 Detoxified Eucalyptus globulus Biomass was dried, milled, and hydrolyzed with 3% H2SO4 (130°C, 1 h) - Lb. pentosus CECT-4023T SHF Batch n.r. 14.5 0.70a 0.28 Moldes et al.2006 Hydrolysate was neutralised with CaCO3 and stirred with 15% w/v of charcoal (room temperature, 1 day) Oak wood chip Biomass was treated with 0.5% H2SO4 (room temperature, overnight) and steam explosion (215°C, 5 h) Commercial cellulase mixture (20 IU/g) supplemented with β-glucosidase (30 IU/g) (50°C, 48 h) Lactobacillus sp. RKY2 SHF Continuous cell recycle (dilution rate 0.16 h−1) n.r. 42 0.95b 6.7 Wee and Ryu, 2009 Recycled paper sludge Biomass was neutralised with 0.3 g HCl/g biomass - Lb. rhamnosus ATCC 7469 SSF Batch n.r. 73 0.97a 2.9 Marques et al.2008 Softwood pre-hydrolysate plus paper mill sludge Softwood particles were sieved and pre-treated with hot water Commercial cellulases (15 FPU/g glucan) plus pectinases (15 mg protein/g mannan) Lactobacillus rhamnosus ATCC-10863 SSF Batch n.r. 60 0.83d 0.62 Shi, Kang and Lee 2015 Sugarcane bagasse Biomass shreds (1–3 mm) were pre-treated with steam and alkali Enzyme preparation from Penicillium janthinellum Lb. delbrueckii mutant Uc-3 SSF Batch L- 67 0.83e 0.93 Adsul, Varmab and Gokhale 2007 Sugarcane bagasse Biomass was dried, milled, treated with 10% NH4OH and hydrolysed with 0.5% HCl (100°C, 5 h). Hydrolysate was detoxified by amberlite treatment - Lc. lactis IO-1 JCM 7638 SHF Batch n.r. 10.9 - 0.14 Laopaiboon et al.2010 Sugarcane bagasse Biomass was ground, sieved and mixed with ionic liquid (120°C, 2 h) Immobilised cellulases (25 FPU/g biomass) from Trichoderma reesei Lactobacillus brevis MTCC 4460 SSF Batch n.r. - 0.52c - Grewal and Khare, 2018 Trimming vine shoots Biomass was dried, milled and hydrolysed with 3% H2SO4 (130°C, 15 min) - Lb. pentosus CECT-4023T SHF Batch n.r. 46 0.78a 0.933 Bustos et al.2005 Trimming vine shoots Substrate was dried, milled and hydrolysed with 3% H2SO4 (130°C, 15 min) - Lb. pentosus CECT-4023T SHF Batch n.r. 24 0.76a 0.51 Moldes et al.2006 Wheat bran Biomass was pre-reduced and sterilised - Lb. amylophilus GV6 Solid state fermentation L- - 0.23c - Naveena et al.2005b Wheat bran Biomass was treated with 1.5% H2SO4 (ratio 1:4 w/v) (80°C, 20 h) - Lb. rhamnosus LA-04–1 SHF Batch L- 75 0.99b 3.75 Li et al.2010b Wheat straw Biomass was ground, sieved and mixed with ionic liquid (120°C, 2 h) Immobilized cellulases (25 FPU/g biomass) from Trichoderma reesei Lactobacillus brevis MTCC 4460 SSF Batch n.r. - 0.49c - Grewal and Khare, 2018 Biomass Physico-chemical treatment(s) Enzymatic treatment Microorganisms Fermentation mode LA enantiomer LA (g/L) Yield Y P/S (g/g) Productivity (g/L/h) Reference Algal cake (de-oiled algal biomass) - Porcine pepsin (37°C, 3 h) plus Lb. casei 12A SHF Batch L- (and traces of D-) 11.17 - - Overbeck, Steele and Broadbent 2016 α-amylase (37°C, 16 h) plus endo-1,4-β-D-glucanase (50°C, 24 h) from Aspergillus niger Barley bran Biomass was dried, milled and hydrolyzed with 3% H2SO4 (130°C, 15 min) - Lb. pentosus CECT-4023T SHF Batch n.r. 33 0.57a 0.60 Moldes et al.2006 Birch wood xylan - Xylanase (1.25 g/L) (60°C, 20 min) Leu. lactis SHO-47 SHF Batch D- 2.3 - - Ohara, Owaki and Sonomoto 2006 Corncob Biomass was dried, milled and hydrolysed with 2% H2SO4 (130°C, 15 min) - Lb. pentosus CECT-4023T SHF Batch n.r. 26 0.53a 0.34 Moldes et al.2006 Corncob Biomass was mashed and hydrolysed with 0.1% H2SO4 (80°C, 1 h) and 0.8% H2SO4 (110°C, 2 h) - Lb. brevis S3F4 SHF Batch n.r. 39.1 0.69a 0.81 Guo et al.2010 Corncob residue - Commercial cellulase mixture (15 FPU/g biomass) Sporolactobacillus inulinus YBS1–5 SHF Fed-Batch D- 107.2 0.85b 1.19 Bai et al.2016 Corn stover Biomass was mashed and hydrolysed with 2% H2SO4 at 10% (w/v) (121°C, 1 h) - Lb. brevis S3F4 SHF Batch n.r. 18.2 0.74a 0.76 Guo et al.2010 Corn stover Biomass was dried, sieved and treated with 5% NaOH (75°C, 3 h) Commercial cellulase, β-glucosidase, and xylanase mixture (30 FPU/g biomass) Lb. pentosus FL0421 SSF Fed-batch n.r. 92.30 0.66c 1.92 Hu et al.2016 Corn stover Biomass was crushed, sieved, dried and treated with 1.5% solid acid (120°C, 80 min) Commercial cellulase mixture (30 FPU/g biomass) Lactobacillus delbrueckii delbrueckii sp. bulgaricus CICC21101 SSF Batch D- 18 - Wang et al.2017 De-oiled cottonseed cake Biomass was ground, sieved and mixed with ionic liquid (120°C, 2 h) Immobilised cellulases (25 FPU/g biomass) from Trichoderma reesei Lactobacillus brevis MTCC 4460 SSF Batch n.r. - 0.22c - Grewal and Khare, 2018 Detoxified Eucalyptus globulus Biomass was dried, milled, and hydrolyzed with 3% H2SO4 (130°C, 1 h) - Lb. pentosus CECT-4023T SHF Batch n.r. 14.5 0.70a 0.28 Moldes et al.2006 Hydrolysate was neutralised with CaCO3 and stirred with 15% w/v of charcoal (room temperature, 1 day) Oak wood chip Biomass was treated with 0.5% H2SO4 (room temperature, overnight) and steam explosion (215°C, 5 h) Commercial cellulase mixture (20 IU/g) supplemented with β-glucosidase (30 IU/g) (50°C, 48 h) Lactobacillus sp. RKY2 SHF Continuous cell recycle (dilution rate 0.16 h−1) n.r. 42 0.95b 6.7 Wee and Ryu, 2009 Recycled paper sludge Biomass was neutralised with 0.3 g HCl/g biomass - Lb. rhamnosus ATCC 7469 SSF Batch n.r. 73 0.97a 2.9 Marques et al.2008 Softwood pre-hydrolysate plus paper mill sludge Softwood particles were sieved and pre-treated with hot water Commercial cellulases (15 FPU/g glucan) plus pectinases (15 mg protein/g mannan) Lactobacillus rhamnosus ATCC-10863 SSF Batch n.r. 60 0.83d 0.62 Shi, Kang and Lee 2015 Sugarcane bagasse Biomass shreds (1–3 mm) were pre-treated with steam and alkali Enzyme preparation from Penicillium janthinellum Lb. delbrueckii mutant Uc-3 SSF Batch L- 67 0.83e 0.93 Adsul, Varmab and Gokhale 2007 Sugarcane bagasse Biomass was dried, milled, treated with 10% NH4OH and hydrolysed with 0.5% HCl (100°C, 5 h). Hydrolysate was detoxified by amberlite treatment - Lc. lactis IO-1 JCM 7638 SHF Batch n.r. 10.9 - 0.14 Laopaiboon et al.2010 Sugarcane bagasse Biomass was ground, sieved and mixed with ionic liquid (120°C, 2 h) Immobilised cellulases (25 FPU/g biomass) from Trichoderma reesei Lactobacillus brevis MTCC 4460 SSF Batch n.r. - 0.52c - Grewal and Khare, 2018 Trimming vine shoots Biomass was dried, milled and hydrolysed with 3% H2SO4 (130°C, 15 min) - Lb. pentosus CECT-4023T SHF Batch n.r. 46 0.78a 0.933 Bustos et al.2005 Trimming vine shoots Substrate was dried, milled and hydrolysed with 3% H2SO4 (130°C, 15 min) - Lb. pentosus CECT-4023T SHF Batch n.r. 24 0.76a 0.51 Moldes et al.2006 Wheat bran Biomass was pre-reduced and sterilised - Lb. amylophilus GV6 Solid state fermentation L- - 0.23c - Naveena et al.2005b Wheat bran Biomass was treated with 1.5% H2SO4 (ratio 1:4 w/v) (80°C, 20 h) - Lb. rhamnosus LA-04–1 SHF Batch L- 75 0.99b 3.75 Li et al.2010b Wheat straw Biomass was ground, sieved and mixed with ionic liquid (120°C, 2 h) Immobilized cellulases (25 FPU/g biomass) from Trichoderma reesei Lactobacillus brevis MTCC 4460 SSF Batch n.r. - 0.49c - Grewal and Khare, 2018 ag of LA/g of total sugar in the hydrolysate. bg of LA/g of glucose in the hydrolysate. cg of LA/g of biomass. dg of LA/g of total hexose sugars. eg of LA/g of cellulose in the biomass. View Large Two main technical challenges are specifically associated with this fermentation strategy: (i) generation of inhibitory compounds by physico-chemical pre-treatment, and (ii) inefficient saccharification of biomass (for an extensive overview see Abdel-Rahman and Sonomoto 2016). Most physico-chemical methods generate inhibitory by-products such as phenolic and furan compounds (e.g. furfural and 5-hydroxymethylfurfural), organic acids (e.g. acetic, formic and levulinic acid) and alcohols (Zhang et al.2016a). The latter may negatively interfere with the activity of cellulolytic/hemicellulolytic enzymes and/or the metabolism of fermenting strains (Abdel-Rahman and Sonomoto 2016). Furthermore, enzymatic hydrolysis of plant polysaccharides frequently suffers from inhibition by end-product (e.g. glucose and cellobiose) accumulation (Abdel-Rahman and Sonomoto 2016). For this reason, separate hydrolysis and fermentation (SHF) approaches can be advantageously replaced by a simultaneous saccharification and fermentation (SSF) strategy. The latter minimises end-product inhibition of hydrolases through rapid consumption of soluble sugars by fermenting microorganisms (Lynd et al.2002). Furthermore, lignocellulose fermentation suffers from the complex nature of this biomass, consisting of different polysaccharides (mainly cellulose, hemicelluloses and pectin) (Lynd et al.2002). Lignocellulose hydrolysis generates sugar mixtures which may undergo inefficient fermentation caused by heterofermentation of pentoses (see the previous section) and/or carbon catabolite repression (Jojima et al.2010). The latter refers to inhibition of pentose metabolism by the presence of glucose leading to non-simultaneous fermentation of sugar mixtures that often leaves most sugar unutilised (Abdel-Rahman and Sonomoto 2016). A wide variety of solutions can be employed to overcome these limitations (Abdel-Rahman, Tashiro and Sonomoto 2011; Abdel-Rahman and Sonomoto 2016). Strategies to reduce the concentration of inhibitory compounds include the choice of alternative milder physico-chemical pre-treatments (e.g. acid or alkaline treatment, steam explosion and ionic liquids) (Abdel-Rahman, Tashiro and Sonomoto 2011) and methods (e.g. chemical additives such as ion-exchange resins and bioabatement) for detoxifying pre-treated biomass (Laopaiboon et al.2010; Jönsson and Martín 2016). Alternatively, the use of enzymes and LAB strains with a higher tolerance to these compounds (either natural or obtained through evolutionary or rational engineering) is a valuable option (Abdel-Rahman and Sonomoto 2016). Cellulase mixtures with different composition and different configurations of the fermentative process (e.g. SHF and SSF) can be used to optimise specific biomass hydrolysis (Abdel-Rahman and Sonomoto 2016). Finally, several LAB strains showing highly efficient metabolism of pentoses are known. Homolactic fermentation of xylose has been observed in Lc. lactis IO-1 (Tanaka et al.2002) or E. faecium QU 50 (Abdel-Rahman et al.2015). Several LAB showing relaxed carbon catabolite repression have been reported. For instance, different Lb. brevis strains were able to simultaneously utilise xylose and glucose derived from hydrolysis of a variety of lignocellulosic feedstocks (Guo et al.2010; Grewal and Khare 2018), while E. faecalis RKY1 co-metabolised mixtures of sucrose, glucose, and/or fructose to LA with high yield (Reddy, Park and Wee 2015) and E. faecium QU 50 homofermentatively utilised glucose/xylose mixtures (Abdel-Rahman et al.2015). Additionally, metabolic engineering strategies have been used to develop strains with improved pentose catabolism, as described in the following sections. Actually, some studies demonstrate that very efficient bioconversion of lignocellulosic biomass into nearly optically pure LA through LAB fermentation (with LA yields close to the theoretical maximum) can be obtained by selecting an optimal combination of pre-treatment, process configuration and microbial strain suitable for a specific substrate (Table 1). However, both the physico-chemical and enzymatic treatments utilised in these studies have significant costs which represent economic barriers at the industrial scale (Okano et al.2010a). Despite extensive research efforts into reducing the cost of the production of cellulases, no significant decrease has been observed since the 1990s (Olson et al.2012). A recent study has estimated the cost of on-site production of cellulases at $10/kg protein (the cost of commercial cellulases is higher) (Klein-Marcuschamer et al.2012). Based on calculations used by Lynd et al. (2017), it can be estimated that the cost of added cellulases per kg of LA produced through lignocellulose fermentation cannot be lower than $0.31. It is worth noting that the cost of fermentative production of LA should be ≤ $0.8/kg for PLA to be economically competitive with fossil fuel-based plastics (Okano et al.2010a). Such an economic target is therefore very challenging through processes such as those described in this section, where the cost of physico-chemical and enzymatic pre-treatment makes the risk significantly too high. Some techno-economic analyses of LA production from renewable biomass have been recently summarised by de Oliveira et al. (2018). Costs may widely vary depending on the process configuration (e.g. the type of feedstocks, method for biomass pre-treatment, LA purification process). In most cases the minimum LA sale price was > $0.8/kg (i.e. between $0.83 and $5/kg). However, a recent study reported a minimum sale price of $0.56/kg for LA produced through fermentation of pre-treated (i.e. dilute acid plus enzymatic hydrolysis) corn stover (Liu et al.2015). Interestingly, cellulase cost was reported as the highest in the entire process (Liu et al.2015). Research for alternative strategies for lignocellulose fermentation with lower dependence on biomass pre-treatment(s) is therefore highly recommended. Significant attention has been dedicated to CBP, i.e. single-pot fermentation of lignocellulosic biomass (Mazzoli 2012). This process configuration differs from SHF and SSF, especially in that it does not involve a dedicated process step for cellulase production (Lynd et al.2005). This could be obtained through cellulolytic microorganisms-LAB consortia or by engineering cellulolytic abilities in LAB. It has been calculated that CBP could lower the cost of biological conversion of lignocellulose by about 78% (Lynd et al.2005). Fermentation of lignocellulosic biomass by cellulolytic microorganisms-LAB consortia Co-cultivation of LAB with native cellulolytic microorganisms could replace saccharification of lignocellulosic biomass by exogenously supplemented cellulases. Utilisation of microbial consortia including cellulolytic strains and high-value compound-producing microbes has been successfully applied to convert cellulosic feedstocks to a variety of products such as ethanol or butanol (Zuroff, Barri Xiques and Curtis 2013; Brethauer and Studer 2014; Wen et al.2014). To date, a single application of this strategy to the production of LA by LAB fermentation has been reported (Shahab et al.2018). In this study, a stable consortium between the cellulolytic fungus Trichoderma reesei and Lb. pentosus based on mutual benefits was developed (Fig. 2). Lb. pentosus efficiently consumes cellobiose thus avoiding inhibition of T. reesei cellulase activity. On the other hand, a by-product of sugar fermentation by Lb. pentosus, i.e. acetic acid, can serve as a carbon source for T. reesei (Shahab et al.2018). Fermentation of whole-slurry pre-treated beech wood by this consortium led to the production of 19.8 g/L of LA through CBP, with an estimated yield of 85.2% of the theoretical maximum (Shahab et al.2018). This study demonstrates that this approach, which mimics microbial syntrophic communities involved in the natural decay of plant material, deserves further investigation. However, difficulties related to the design and maintenance of stable artificial microbial communities represent the main challenges to this strategy (Johns et al.2016). Figure 2. View largeDownload slide Schematic representation of T. reesei/Lb. pentosus consortium developed by Shahab et al. (2018). T. reesei grows as a biofilm on the surface of an oxygen permeable membrane and secretes cellulases and hemicellulases (EGI: endoglucanase I, CBHI: cellobiohydrolase I, CBHII: cellobiohydrolase II, BXL: β-xylosidase, XLN: β-endoxylanase). Soluble saccharides produced by T. reesei enzymes are fermented by Lb. pentosus to lactic and acetic acid. Acetic acid can serve as energy source for T. reesei (modified from Shahab et al.2018). Figure 2. View largeDownload slide Schematic representation of T. reesei/Lb. pentosus consortium developed by Shahab et al. (2018). T. reesei grows as a biofilm on the surface of an oxygen permeable membrane and secretes cellulases and hemicellulases (EGI: endoglucanase I, CBHI: cellobiohydrolase I, CBHII: cellobiohydrolase II, BXL: β-xylosidase, XLN: β-endoxylanase). Soluble saccharides produced by T. reesei enzymes are fermented by Lb. pentosus to lactic and acetic acid. Acetic acid can serve as energy source for T. reesei (modified from Shahab et al.2018). Construction of recombinant cellulolytic/hemicellulolytic LAB through metabolic engineering: the state of the art and future directions Research at the forefront of the development of second generation biorefinery processes includes endowing microorganisms that produce high-value chemicals with the ability to directly ferment lignocellulose without prior physico-chemical and/or enzymatic pre-treatment through recombinant techniques (Mazzoli, Lamberti and Pessione 2012). The number of examples of recombinant cellulolytic strategies (RCS) addressing LAB is growing (Mazzoli et al.2014; Gandini et al.2017; Stern et al.2018). The natural ability to grow on lignocellulose relies on multiple-enzyme systems that mainly consist of glycosyl hydrolases and polysaccharide lyases (Lynd et al.2002). Most studies have addressed to two main paradigms for cellulose depolymerisation, the non-complex enzyme model of aerobic fungi and bacteria, and the cellulosome complexes of anaerobic microorganisms (Lynd et al.2002). The latter are based on scaffolding proteins (i.e. scaffoldins) that generally provide multiple functions, i.e. the ability to bind enzyme subunits (thus organising the enzyme complex architecture), polysaccharides and cell surface through specific protein domains (Mazzoli, Lamberti and Pessione 2012). RCS aim at mimicking nature by engineering minimal cellulolytic systems (Mazzoli 2012) (Fig. 3). Traditionally, a minimal non-complex system able to act efficiently on cellulosic substrates consists of an exoglucanase, an endoglucanase and a β-glucosidase (Lynd et al.2002) (Fig. 3A, B). A mini-scaffoldin is also required in the case of mini- or designer-cellulosomes (Fig. 3C). However, in most studies aimed at LAB engineering with heterologous cellulases reported to date, a single cellulase was introduced (for an extensive review see Mazzoli et al.2014) (Table 2). This modification may enable metabolisation of short cellodextrins, or partial hydrolysis of cellulose/hemicellulose, but is insufficient for these recombinant strains to efficiently grow on and ferment complex lignocellulosic substrates (Mazzoli et al.2014) (Table 2). Actually, most of these recombinant strains were intended to be used as inoculants for silage fermentation (i.e. for improving silage acidification and/or digestibility) (Bates et al.1989; Scheirlinck, Mahillon and Joos 1989; Rossi et al.2001; Ozkose et al.2009) rather than as biocatalysts in biorefinery processes. More recently, construction of cellulolytic LAB for the industrial production of LA has been considered. Among the most performant strains, Lb. plantarum engineered with Cel8A endoglucanase from C. thermocellum was able to grow on cellooligosaccharides up to 5–6 glucose residues long (Okano et al.2010b). Several studies have reported that expression of heterologous cellulases may be toxic (Mingardon et al.2011; Moraïs et al.2014). Hence, expression of multiple cellulases is extremely challenging. The development of artificial syntrophic consortia (consisting of recombinant strains that biosynthesise single different cellulase-system components) have been used to circumvent this bottleneck (Moraïs et al.2013; 2014; Stern et al.2018). Morais and co-workers (2013) have shown the potential of simple consortia of recombinant Lb. plantarum strains secreting cellulase-xylanase mixtures for biomass (i.e. wheat straw) bioconversion. The same research group has significantly improved its hemi/cellulolytic LAB consortium over time by including strains that biosynthesise different (i) surface-anchored mini-scaffoldins (each able to bind up to four enzymatic subunits), (ii) adaptor mini-scaffoldins (each able to bind up to two enzymatic subunits), and (iii) endoglucanases and xylanases (Moraïs et al.2014; Stern et al.2018) (Fig. 3C). Synthetic Lb. plantarum consortia that display mini-cellulosomes incorporating up to six enzymatic subunits could be developed, which would be a remarkable result (Stern et al.2018). Although these enzyme complexes showed improved hydrolysis of wheat straw, they were unable to support growth of Lb. plantarum on wheat straw as the sole carbon source. This result is most likely related to the amount and/or type of sugars released by the specific designer cellulosomes which seems insufficient/unsuitable for Lb. plantarum growth (Stern et al.2018). Additionally, it has to be remembered that the management of these consortia at the industrial scale may not be trivial. Recently, a cellulase system consisting of a β-glucosidase and an endoglucanase was engineered in a single Lc. lactis strain through the construction of an artificial operon (Gandini et al.2017). This strain could directly convert cellooligosaccharides up to at least cellooctaose to L-LA with high yield. However, the basal expression triggered by the promoter used (P32) was shown to be not very high, and further improvement of this strain for application in biorefinery will be required, e.g. through increased cellulase expression (Gandini et al.2017). Figure 3. View largeDownload slide Paradigms for recombinant cellulolytic strategies reported in LAB. Recombinant cells (A) secreting minimal non-complex cellulase system, biosynthesising (B) surface-displayed cellulases, or (C) surface-displayed designer cellulosomes are depicted. Bgl, β-glucosidase; Eng, endoglucanase; Exg, exoglucanase. Figure 3. View largeDownload slide Paradigms for recombinant cellulolytic strategies reported in LAB. Recombinant cells (A) secreting minimal non-complex cellulase system, biosynthesising (B) surface-displayed cellulases, or (C) surface-displayed designer cellulosomes are depicted. Bgl, β-glucosidase; Eng, endoglucanase; Exg, exoglucanase. Table 2. Examples of recombinant cellulolytic strategies (RCS) on lactic acid bacteria (LAB). Recombinant LAB strains were engineered with heterologous cellulase/hemicellulose systems. Lb., Lactobacillus; Lc., Lactococcus. Strain(s) Heterologous protein(s) expressed Transcriptional promoter Gene cloning strategy Heterologous protein expression/secretion levela Improved phenotypic properties of the strain Reference Lb. gasseri ATCC 33323 Ce8lA endoglucanase from Clostridium thermocellum Inducible (lacA promoter) Plasmid 722 U/L (CMC)b Hydrolysis of CMC Cho, Choi and Chung 2000 Lb. jonhsonii NCK 88 Cel8A endoglucanase from C. thermocellum Inducible (lacA promoter) Plasmid 759 U/L (CMC)b Hydrolysis of CMC Cho, Choi and Chung 2000 Lb. plantarum strains B41 and Lp80 Cel8A cellulase from Bacillus sp. N-4 Not indicated Chromosome integration 34.24/43.61 U/L (CMC)b Increased silage acidification Rossi et al.2001 Lb. plantarum Lp80 Cel8A endoglucanase from C. thermocellum Not indicated Chromosome integration ≈ 90 U/L (CMC)b Hydrolysis of CMC Scheirlinck, Mahillon and Joos 1989 Lb. plantarum NCDO 1193 Cel5E endoglucanase from C. thermocellum Not indicated Plasmid 1996 U/L (CMC)b Hydrolysis of CMC Bates et al.1989 Lb. plantarum NCIMB 8826 (Δldh1) Cel8A endoglucanase from C. thermocellum Constitutive (ClpC core promoter) Plasmid 6.03 U/L (barley β-glucan)b Growth on cellohexaose Okano et al.2010b Lb. plantarum WCFS1 Cel6A endoglucanase from Thermobifida fusca Inducible (sakacin P promoter) Plasmid 280 U/L (PASC)b Hydrolysis of sodium hypochlorite-pre-treated wheat straw Morais et al.2013 Lb. plantarum WCFS1 Xyn11A endoxylanase from T. fusca Inducible (sakacin P promoter) Plasmid 3360 U/L (oat spelt xylan)b Hydrolysis of sodium hypochlorite-pre-treated wheat straw Morais et al.2013 Lb. plantarum WCFS1 Cel6A endoglucanase plus Xyn11A endoxylanase from T. fusca plus chimeric scaffoldin-AT (synthetic consortium) Inducible (sakacin P promoter) Plasmid Hydrolysis of sodium hypochlorite-pre-treated wheat straw Morais et al.2014 Lb. plantarum WCFS1 Chimeric GH5 and GH9 endoglucanases and GH10 and GH11 xylanases from Clostridium papyrosolvens plus chimeric adaptor and anchoring scaffoldins (synthetic consortium) Inducible (sakacin P promoter) Plasmid 0.2–59.1 nMb,c Hydrolysis of sodium hypochlorite-pre-treated wheat straw Stern et al.2018 Strain(s) Heterologous protein(s) expressed Transcriptional promoter Gene cloning strategy Heterologous protein expression/secretion levela Improved phenotypic properties of the strain Reference Lb. gasseri ATCC 33323 Ce8lA endoglucanase from Clostridium thermocellum Inducible (lacA promoter) Plasmid 722 U/L (CMC)b Hydrolysis of CMC Cho, Choi and Chung 2000 Lb. jonhsonii NCK 88 Cel8A endoglucanase from C. thermocellum Inducible (lacA promoter) Plasmid 759 U/L (CMC)b Hydrolysis of CMC Cho, Choi and Chung 2000 Lb. plantarum strains B41 and Lp80 Cel8A cellulase from Bacillus sp. N-4 Not indicated Chromosome integration 34.24/43.61 U/L (CMC)b Increased silage acidification Rossi et al.2001 Lb. plantarum Lp80 Cel8A endoglucanase from C. thermocellum Not indicated Chromosome integration ≈ 90 U/L (CMC)b Hydrolysis of CMC Scheirlinck, Mahillon and Joos 1989 Lb. plantarum NCDO 1193 Cel5E endoglucanase from C. thermocellum Not indicated Plasmid 1996 U/L (CMC)b Hydrolysis of CMC Bates et al.1989 Lb. plantarum NCIMB 8826 (Δldh1) Cel8A endoglucanase from C. thermocellum Constitutive (ClpC core promoter) Plasmid 6.03 U/L (barley β-glucan)b Growth on cellohexaose Okano et al.2010b Lb. plantarum WCFS1 Cel6A endoglucanase from Thermobifida fusca Inducible (sakacin P promoter) Plasmid 280 U/L (PASC)b Hydrolysis of sodium hypochlorite-pre-treated wheat straw Morais et al.2013 Lb. plantarum WCFS1 Xyn11A endoxylanase from T. fusca Inducible (sakacin P promoter) Plasmid 3360 U/L (oat spelt xylan)b Hydrolysis of sodium hypochlorite-pre-treated wheat straw Morais et al.2013 Lb. plantarum WCFS1 Cel6A endoglucanase plus Xyn11A endoxylanase from T. fusca plus chimeric scaffoldin-AT (synthetic consortium) Inducible (sakacin P promoter) Plasmid Hydrolysis of sodium hypochlorite-pre-treated wheat straw Morais et al.2014 Lb. plantarum WCFS1 Chimeric GH5 and GH9 endoglucanases and GH10 and GH11 xylanases from Clostridium papyrosolvens plus chimeric adaptor and anchoring scaffoldins (synthetic consortium) Inducible (sakacin P promoter) Plasmid 0.2–59.1 nMb,c Hydrolysis of sodium hypochlorite-pre-treated wheat straw Stern et al.2018 View Large Table 2. Examples of recombinant cellulolytic strategies (RCS) on lactic acid bacteria (LAB). Recombinant LAB strains were engineered with heterologous cellulase/hemicellulose systems. Lb., Lactobacillus; Lc., Lactococcus. Strain(s) Heterologous protein(s) expressed Transcriptional promoter Gene cloning strategy Heterologous protein expression/secretion levela Improved phenotypic properties of the strain Reference Lb. gasseri ATCC 33323 Ce8lA endoglucanase from Clostridium thermocellum Inducible (lacA promoter) Plasmid 722 U/L (CMC)b Hydrolysis of CMC Cho, Choi and Chung 2000 Lb. jonhsonii NCK 88 Cel8A endoglucanase from C. thermocellum Inducible (lacA promoter) Plasmid 759 U/L (CMC)b Hydrolysis of CMC Cho, Choi and Chung 2000 Lb. plantarum strains B41 and Lp80 Cel8A cellulase from Bacillus sp. N-4 Not indicated Chromosome integration 34.24/43.61 U/L (CMC)b Increased silage acidification Rossi et al.2001 Lb. plantarum Lp80 Cel8A endoglucanase from C. thermocellum Not indicated Chromosome integration ≈ 90 U/L (CMC)b Hydrolysis of CMC Scheirlinck, Mahillon and Joos 1989 Lb. plantarum NCDO 1193 Cel5E endoglucanase from C. thermocellum Not indicated Plasmid 1996 U/L (CMC)b Hydrolysis of CMC Bates et al.1989 Lb. plantarum NCIMB 8826 (Δldh1) Cel8A endoglucanase from C. thermocellum Constitutive (ClpC core promoter) Plasmid 6.03 U/L (barley β-glucan)b Growth on cellohexaose Okano et al.2010b Lb. plantarum WCFS1 Cel6A endoglucanase from Thermobifida fusca Inducible (sakacin P promoter) Plasmid 280 U/L (PASC)b Hydrolysis of sodium hypochlorite-pre-treated wheat straw Morais et al.2013 Lb. plantarum WCFS1 Xyn11A endoxylanase from T. fusca Inducible (sakacin P promoter) Plasmid 3360 U/L (oat spelt xylan)b Hydrolysis of sodium hypochlorite-pre-treated wheat straw Morais et al.2013 Lb. plantarum WCFS1 Cel6A endoglucanase plus Xyn11A endoxylanase from T. fusca plus chimeric scaffoldin-AT (synthetic consortium) Inducible (sakacin P promoter) Plasmid Hydrolysis of sodium hypochlorite-pre-treated wheat straw Morais et al.2014 Lb. plantarum WCFS1 Chimeric GH5 and GH9 endoglucanases and GH10 and GH11 xylanases from Clostridium papyrosolvens plus chimeric adaptor and anchoring scaffoldins (synthetic consortium) Inducible (sakacin P promoter) Plasmid 0.2–59.1 nMb,c Hydrolysis of sodium hypochlorite-pre-treated wheat straw Stern et al.2018 Strain(s) Heterologous protein(s) expressed Transcriptional promoter Gene cloning strategy Heterologous protein expression/secretion levela Improved phenotypic properties of the strain Reference Lb. gasseri ATCC 33323 Ce8lA endoglucanase from Clostridium thermocellum Inducible (lacA promoter) Plasmid 722 U/L (CMC)b Hydrolysis of CMC Cho, Choi and Chung 2000 Lb. jonhsonii NCK 88 Cel8A endoglucanase from C. thermocellum Inducible (lacA promoter) Plasmid 759 U/L (CMC)b Hydrolysis of CMC Cho, Choi and Chung 2000 Lb. plantarum strains B41 and Lp80 Cel8A cellulase from Bacillus sp. N-4 Not indicated Chromosome integration 34.24/43.61 U/L (CMC)b Increased silage acidification Rossi et al.2001 Lb. plantarum Lp80 Cel8A endoglucanase from C. thermocellum Not indicated Chromosome integration ≈ 90 U/L (CMC)b Hydrolysis of CMC Scheirlinck, Mahillon and Joos 1989 Lb. plantarum NCDO 1193 Cel5E endoglucanase from C. thermocellum Not indicated Plasmid 1996 U/L (CMC)b Hydrolysis of CMC Bates et al.1989 Lb. plantarum NCIMB 8826 (Δldh1) Cel8A endoglucanase from C. thermocellum Constitutive (ClpC core promoter) Plasmid 6.03 U/L (barley β-glucan)b Growth on cellohexaose Okano et al.2010b Lb. plantarum WCFS1 Cel6A endoglucanase from Thermobifida fusca Inducible (sakacin P promoter) Plasmid 280 U/L (PASC)b Hydrolysis of sodium hypochlorite-pre-treated wheat straw Morais et al.2013 Lb. plantarum WCFS1 Xyn11A endoxylanase from T. fusca Inducible (sakacin P promoter) Plasmid 3360 U/L (oat spelt xylan)b Hydrolysis of sodium hypochlorite-pre-treated wheat straw Morais et al.2013 Lb. plantarum WCFS1 Cel6A endoglucanase plus Xyn11A endoxylanase from T. fusca plus chimeric scaffoldin-AT (synthetic consortium) Inducible (sakacin P promoter) Plasmid Hydrolysis of sodium hypochlorite-pre-treated wheat straw Morais et al.2014 Lb. plantarum WCFS1 Chimeric GH5 and GH9 endoglucanases and GH10 and GH11 xylanases from Clostridium papyrosolvens plus chimeric adaptor and anchoring scaffoldins (synthetic consortium) Inducible (sakacin P promoter) Plasmid 0.2–59.1 nMb,c Hydrolysis of sodium hypochlorite-pre-treated wheat straw Stern et al.2018 View Large Table 2. (continued) Strain(s) Heterologous protein(s) expressed Transcriptional promoter Gene cloning strategy Heterologous protein expression/secretion levela Improved phenotypic properties of the strain Reference Lb. reuteri XC1 CelW endoglucanase from Bacillus subtilis WL001 and phyW phytase from Aspergillus fumigatus WL002 (artificial operon) Constitutive (LdhL promoter) Plasmid 960 U/L (CMC)b Hydrolysis of CMC Wang et al.2014 Lc. lactis HtrA NZ9000 Fragments of CipA scaffoldin from C. thermocellum Inducible (nisA promoter) Plasmid 9 × 103 scaffolds/celld Scaffoldins displayed on the cell surface Wieckzoreck and Martin, 2010 Lc. lactis IL1403 BglA β-glucan glucohydrolase and EngD Endoglucanase/Xylanase from Clostridium cellulovorans (artificial operon) Constitutive (P32 promoter) Plasmid 1.220 U/L (pNGP)b; 157 U/L (Azo-CMC)b Hydrolysis of CMC; Growth on cellooctaose Gandini et al.2017 Lc. lactis strains IL1403 and MG1363 Cellulase from Neocallimastix sp. Inducible (lacZ promoter) Plasmid 5.9 U (CMC)b,e Hydrolysis of CMC Ozkose et al.2009 Lc. lactis MG1316 Xylanase from Bacillus coagulans ST-6 Constitutive (P32 promoter) Plasmid ≈87 U/L (xylan)c Hydrolysis of RBB-xylan Raha et al.2006 Lc. lactis MG1316 Egl3 endoglucanase from Trichoderma reesei Constitutive (P32 promoter) Plasmid 1118 U/L (CMC)b Improved metabolisation of paper and wheat straw Liu et al 2017 Strain(s) Heterologous protein(s) expressed Transcriptional promoter Gene cloning strategy Heterologous protein expression/secretion levela Improved phenotypic properties of the strain Reference Lb. reuteri XC1 CelW endoglucanase from Bacillus subtilis WL001 and phyW phytase from Aspergillus fumigatus WL002 (artificial operon) Constitutive (LdhL promoter) Plasmid 960 U/L (CMC)b Hydrolysis of CMC Wang et al.2014 Lc. lactis HtrA NZ9000 Fragments of CipA scaffoldin from C. thermocellum Inducible (nisA promoter) Plasmid 9 × 103 scaffolds/celld Scaffoldins displayed on the cell surface Wieckzoreck and Martin, 2010 Lc. lactis IL1403 BglA β-glucan glucohydrolase and EngD Endoglucanase/Xylanase from Clostridium cellulovorans (artificial operon) Constitutive (P32 promoter) Plasmid 1.220 U/L (pNGP)b; 157 U/L (Azo-CMC)b Hydrolysis of CMC; Growth on cellooctaose Gandini et al.2017 Lc. lactis strains IL1403 and MG1363 Cellulase from Neocallimastix sp. Inducible (lacZ promoter) Plasmid 5.9 U (CMC)b,e Hydrolysis of CMC Ozkose et al.2009 Lc. lactis MG1316 Xylanase from Bacillus coagulans ST-6 Constitutive (P32 promoter) Plasmid ≈87 U/L (xylan)c Hydrolysis of RBB-xylan Raha et al.2006 Lc. lactis MG1316 Egl3 endoglucanase from Trichoderma reesei Constitutive (P32 promoter) Plasmid 1118 U/L (CMC)b Improved metabolisation of paper and wheat straw Liu et al 2017 aMaximum values reported in each study. Substrates used for determining enzyme activity are indicated in parentheses. Azo-CMC, carboxy methyl cellulose; N3-G5-β-CNP, 2-chloro-4-nitrophenyl-65-azido-65-deoxy-β-maltopentaoside; PASC, phosphoric acid-swollen cellulose; pNGP, p-nitrophenyl-b-D-glucopyranoside (pNGP); RBB-xylan, remazol brilliant blue xylan. bEnzyme activity/protein quantification measured in extracellular fraction. cProtein quantification through ELISA-based binding assays on cultures with OD600nm=1. dProteins displayed on the cell surface. eThe volume of extracellular extract used in this study was not reported. View Large Table 2. (continued) Strain(s) Heterologous protein(s) expressed Transcriptional promoter Gene cloning strategy Heterologous protein expression/secretion levela Improved phenotypic properties of the strain Reference Lb. reuteri XC1 CelW endoglucanase from Bacillus subtilis WL001 and phyW phytase from Aspergillus fumigatus WL002 (artificial operon) Constitutive (LdhL promoter) Plasmid 960 U/L (CMC)b Hydrolysis of CMC Wang et al.2014 Lc. lactis HtrA NZ9000 Fragments of CipA scaffoldin from C. thermocellum Inducible (nisA promoter) Plasmid 9 × 103 scaffolds/celld Scaffoldins displayed on the cell surface Wieckzoreck and Martin, 2010 Lc. lactis IL1403 BglA β-glucan glucohydrolase and EngD Endoglucanase/Xylanase from Clostridium cellulovorans (artificial operon) Constitutive (P32 promoter) Plasmid 1.220 U/L (pNGP)b; 157 U/L (Azo-CMC)b Hydrolysis of CMC; Growth on cellooctaose Gandini et al.2017 Lc. lactis strains IL1403 and MG1363 Cellulase from Neocallimastix sp. Inducible (lacZ promoter) Plasmid 5.9 U (CMC)b,e Hydrolysis of CMC Ozkose et al.2009 Lc. lactis MG1316 Xylanase from Bacillus coagulans ST-6 Constitutive (P32 promoter) Plasmid ≈87 U/L (xylan)c Hydrolysis of RBB-xylan Raha et al.2006 Lc. lactis MG1316 Egl3 endoglucanase from Trichoderma reesei Constitutive (P32 promoter) Plasmid 1118 U/L (CMC)b Improved metabolisation of paper and wheat straw Liu et al 2017 Strain(s) Heterologous protein(s) expressed Transcriptional promoter Gene cloning strategy Heterologous protein expression/secretion levela Improved phenotypic properties of the strain Reference Lb. reuteri XC1 CelW endoglucanase from Bacillus subtilis WL001 and phyW phytase from Aspergillus fumigatus WL002 (artificial operon) Constitutive (LdhL promoter) Plasmid 960 U/L (CMC)b Hydrolysis of CMC Wang et al.2014 Lc. lactis HtrA NZ9000 Fragments of CipA scaffoldin from C. thermocellum Inducible (nisA promoter) Plasmid 9 × 103 scaffolds/celld Scaffoldins displayed on the cell surface Wieckzoreck and Martin, 2010 Lc. lactis IL1403 BglA β-glucan glucohydrolase and EngD Endoglucanase/Xylanase from Clostridium cellulovorans (artificial operon) Constitutive (P32 promoter) Plasmid 1.220 U/L (pNGP)b; 157 U/L (Azo-CMC)b Hydrolysis of CMC; Growth on cellooctaose Gandini et al.2017 Lc. lactis strains IL1403 and MG1363 Cellulase from Neocallimastix sp. Inducible (lacZ promoter) Plasmid 5.9 U (CMC)b,e Hydrolysis of CMC Ozkose et al.2009 Lc. lactis MG1316 Xylanase from Bacillus coagulans ST-6 Constitutive (P32 promoter) Plasmid ≈87 U/L (xylan)c Hydrolysis of RBB-xylan Raha et al.2006 Lc. lactis MG1316 Egl3 endoglucanase from Trichoderma reesei Constitutive (P32 promoter) Plasmid 1118 U/L (CMC)b Improved metabolisation of paper and wheat straw Liu et al 2017 aMaximum values reported in each study. Substrates used for determining enzyme activity are indicated in parentheses. Azo-CMC, carboxy methyl cellulose; N3-G5-β-CNP, 2-chloro-4-nitrophenyl-65-azido-65-deoxy-β-maltopentaoside; PASC, phosphoric acid-swollen cellulose; pNGP, p-nitrophenyl-b-D-glucopyranoside (pNGP); RBB-xylan, remazol brilliant blue xylan. bEnzyme activity/protein quantification measured in extracellular fraction. cProtein quantification through ELISA-based binding assays on cultures with OD600nm=1. dProteins displayed on the cell surface. eThe volume of extracellular extract used in this study was not reported. View Large Attempts to improve hemicellulose metabolism in LAB include several examples of expression of heterologous xylanases (Raha et al.2006; Morais et al.2013; Gandini et al.2017) (Table 3). Morais et al. (2013) demonstrated that xylanase-expressing Lb. plantarum improved cellulose accessibility. Most other metabolic engineering studies have concerned the improvement of pentose conversion into LA through disruption of the phosphoketolase pathway and introduction or enhancement of the pentose phosphate pathway (Okano et al 2009a; Shinkawa et al.2011; Qiu, Gao and Bao 2018) (Table 2). These studies obtained impressive results since engineered strains were nearly able to achieve homolactic fermentation of xylose and/or arabinose (Table 3). In addition, some engineered strains showed the ability to co-ferment glucose/xylose mixtures without carbon catabolite repression (Yoshida et al.2011; Zhang et al.2016b). Table 3. Recombinant LAB showing improved pentose metabolism. Tkt, transketolase; XylA, xylose isomerase; XylB, xylulose kinase. Strain Heterologous protein(s) expressed Transcriptional promoter Gene cloning strategy Improved phenotypic properties of the strain Reference Lb. plantarum NCIMB 8826 (Δldh1-xpk1) Tkt from Lc. lactis IL1403 (replacing endogenous phosphoketolase Xpk1) Not indicated Chromosome integration Almost homolactic (D-LA) fermentation of arabinose Okano et al.2009a Lb. plantarum NCIMB 8826 (Δldh1-xpk1-xpk2) Tkt from Lc. lactis IL1403 (replacing endogenous phosphoketolase Xpk1 and Xpk2); XylA and XylB from Lb. pentosus NRIC 1069 Not indicated Chromosome integration Almost homolactic (D-LA) fermentation of xylose Okano et al.2009b Lc. lactis IL1403 (Δpkt) XylA and XylB from Lc. lactis IO-1 and endogenous tkt replacing endogenous phosphoketolase (pkt) Inducible (xylose) for XylAB. Not indicated for tkt Plasmid and chromosome integration Almost homolactic (L-LA) fermentation of xylose Shinkawa et al.2011 Ped. acidilactici TY112 (ΔldhD-pkt-ackA2) Transaldolase, tkt (replacing endogenous phosphoketolase, pkt), XylA and XylB (replacing endogenous acetate kinase, ackA2) from Pediococcus acidilactici DSM20284 Constitutive (PldhD) Chromosome integration Almost homolactic (L-LA) fermentation of xylose Qiu, Gao and Bao 2018 Strain Heterologous protein(s) expressed Transcriptional promoter Gene cloning strategy Improved phenotypic properties of the strain Reference Lb. plantarum NCIMB 8826 (Δldh1-xpk1) Tkt from Lc. lactis IL1403 (replacing endogenous phosphoketolase Xpk1) Not indicated Chromosome integration Almost homolactic (D-LA) fermentation of arabinose Okano et al.2009a Lb. plantarum NCIMB 8826 (Δldh1-xpk1-xpk2) Tkt from Lc. lactis IL1403 (replacing endogenous phosphoketolase Xpk1 and Xpk2); XylA and XylB from Lb. pentosus NRIC 1069 Not indicated Chromosome integration Almost homolactic (D-LA) fermentation of xylose Okano et al.2009b Lc. lactis IL1403 (Δpkt) XylA and XylB from Lc. lactis IO-1 and endogenous tkt replacing endogenous phosphoketolase (pkt) Inducible (xylose) for XylAB. Not indicated for tkt Plasmid and chromosome integration Almost homolactic (L-LA) fermentation of xylose Shinkawa et al.2011 Ped. acidilactici TY112 (ΔldhD-pkt-ackA2) Transaldolase, tkt (replacing endogenous phosphoketolase, pkt), XylA and XylB (replacing endogenous acetate kinase, ackA2) from Pediococcus acidilactici DSM20284 Constitutive (PldhD) Chromosome integration Almost homolactic (L-LA) fermentation of xylose Qiu, Gao and Bao 2018 View Large Table 3. Recombinant LAB showing improved pentose metabolism. Tkt, transketolase; XylA, xylose isomerase; XylB, xylulose kinase. Strain Heterologous protein(s) expressed Transcriptional promoter Gene cloning strategy Improved phenotypic properties of the strain Reference Lb. plantarum NCIMB 8826 (Δldh1-xpk1) Tkt from Lc. lactis IL1403 (replacing endogenous phosphoketolase Xpk1) Not indicated Chromosome integration Almost homolactic (D-LA) fermentation of arabinose Okano et al.2009a Lb. plantarum NCIMB 8826 (Δldh1-xpk1-xpk2) Tkt from Lc. lactis IL1403 (replacing endogenous phosphoketolase Xpk1 and Xpk2); XylA and XylB from Lb. pentosus NRIC 1069 Not indicated Chromosome integration Almost homolactic (D-LA) fermentation of xylose Okano et al.2009b Lc. lactis IL1403 (Δpkt) XylA and XylB from Lc. lactis IO-1 and endogenous tkt replacing endogenous phosphoketolase (pkt) Inducible (xylose) for XylAB. Not indicated for tkt Plasmid and chromosome integration Almost homolactic (L-LA) fermentation of xylose Shinkawa et al.2011 Ped. acidilactici TY112 (ΔldhD-pkt-ackA2) Transaldolase, tkt (replacing endogenous phosphoketolase, pkt), XylA and XylB (replacing endogenous acetate kinase, ackA2) from Pediococcus acidilactici DSM20284 Constitutive (PldhD) Chromosome integration Almost homolactic (L-LA) fermentation of xylose Qiu, Gao and Bao 2018 Strain Heterologous protein(s) expressed Transcriptional promoter Gene cloning strategy Improved phenotypic properties of the strain Reference Lb. plantarum NCIMB 8826 (Δldh1-xpk1) Tkt from Lc. lactis IL1403 (replacing endogenous phosphoketolase Xpk1) Not indicated Chromosome integration Almost homolactic (D-LA) fermentation of arabinose Okano et al.2009a Lb. plantarum NCIMB 8826 (Δldh1-xpk1-xpk2) Tkt from Lc. lactis IL1403 (replacing endogenous phosphoketolase Xpk1 and Xpk2); XylA and XylB from Lb. pentosus NRIC 1069 Not indicated Chromosome integration Almost homolactic (D-LA) fermentation of xylose Okano et al.2009b Lc. lactis IL1403 (Δpkt) XylA and XylB from Lc. lactis IO-1 and endogenous tkt replacing endogenous phosphoketolase (pkt) Inducible (xylose) for XylAB. Not indicated for tkt Plasmid and chromosome integration Almost homolactic (L-LA) fermentation of xylose Shinkawa et al.2011 Ped. acidilactici TY112 (ΔldhD-pkt-ackA2) Transaldolase, tkt (replacing endogenous phosphoketolase, pkt), XylA and XylB (replacing endogenous acetate kinase, ackA2) from Pediococcus acidilactici DSM20284 Constitutive (PldhD) Chromosome integration Almost homolactic (L-LA) fermentation of xylose Qiu, Gao and Bao 2018 View Large Although the number of RCS targeted to LAB engineering is growing, progress in research on these organisms is still far behind that achieved in other microbial models such as S. cerevisiae. All of the aforementioned examples suffer from multiple limits which hamper the application of such recombinant LAB to industrial fermentation of real cellulosic substrates. In most cases, inducible promoters have been used to control the transcription of heterologous cellulases (Table 2). Inducible promoters have been preferred so as to delay cellulase expression in the late exponential phase, thus avoiding major growth inhibition by cellulase expression. However, utilisation of inducible promoters is not financially sustainable at the industrial scale, since large amounts of expensive inducer would be employed. A further problem may be represented by the limited amount of cellulases which are secreted by the recombinant cellulolytic LAB obtained to date (Table 2) (Mazzoli et al.2014). As a basis for comparison, cellulase activity of native cellulosome-producing Clostridium thermocellum on cellulosic substrates can range between 100–1000 U/L (Krauss, Zverlov and Schwarz 2012; You et al.2012). In many state-of-the-art recombinant cellulolytic LAB, measured cellulolytic activities are around or under the lower end of this range (Table 2) and are strongly dependent on specific cellulase (Stern et al.2018). Although available genetic tools for LAB are relatively abundant, those enabling strong constitutive expression of proteins have for a long time been restricted to a few choices, such as the lactococcal P32 and P45 promoters (Table 2). Fortunately, new constitutive promoters with different strengths have been discovered for both Lactococci (Zhu et al.2015) and Lactobacilli (Tauer et al.; 2014; Duong et al.2011). Alternatively, the generation of libraries of synthetic constitutive promoters displaying a wide range of strength (Jensen and Hammer 1998; Rud et al.2006) appears to be a potent tool for mimicking native cellulase systems, in which the highest synergism is obtained for non-equimolar expression of different enzymes (Mazzoli, Lamberti and Pessione 2012). Additional tools to increase cellulase/hemicellulose expression in LAB include improvement of mRNA stability (Narita, Ishida and Okano 2006; Okano et al.2010a) or increase of translation efficiency through design of synthetic genes with optimised codon usage (Johnston et al.2014; Dong et al.2015; Li et al.2016). The most challenging factor in heterologous expression of cellulases consists in finding an efficient secretion strategy (Mazzoli, Lamberti and Pessione 2012). Saturation of transmembrane transport mechanisms of the host and accumulation of misfolded or aggregated proteins is the most probable factor causing toxicity of heterologous cellulases (Illmen et al.2011; Morais et al.2014). Mechanisms of cellulase secretion in native cellulolytic microorganisms are almost completely unknown. Based on analysis of signal peptide sequences, a recent study postulated that only about 6% of the known cellulase is secreted through established mechanisms (e.g. the Sec or Tat pathway) (Yan and Wu 2014). In this scenario, studies on heterologous cellulase expression have often been based on a trial-and-error approach in order to find enzymes compatible with the host (Illmen et al.2011; Mingardon et al.2011). Luckily, mechanisms of protein secretion in cellulolytic clostridia and LAB have shown some similarities since a number of components of cellulase systems of clostridia, with their original signal peptide, could be efficiently secreted by Lb. plantarum or Lc. lactis (Wieczoreck and Martin 2010; Okano et al.2010b; Morais et al.2013; Gandini et al.2017). Alternatively, original signal peptides of cellulases can be replaced with sequences (i.e. signal peptides, propeptides) promoting efficient protein secretion in the host of interest (Dong et al.2015; Lim et al.2017). Typically, the native (or engineered) signal peptide of Usp45, the main secreted protein of Lc. lactis, has been used for promoting the secretion of heterologous proteins in Lc. lactis (Morello et al.2008; Ng and Sarkar 2013), including cellulase system components from different microorganisms (Wieczoreck and Martin 2010; Wang et al.2014; Liu et al.2017), while Lp3050 or Lp2588 leader peptides have been used to enable secretion of cellulosomal components in Lb. plantarum (Stern et al.2018). All these tools can significantly help development of RCS of LAB; however, they cannot guarantee their success because currently it still largely depends on specific protein/host combinations. Signal peptides and propeptides most likely play additional roles in protein translocation, maturation and folding, which needs better understanding (Harwood and Cranenburgh 2008; Mazzoli, Lamberti and Pessione 2012; Yan and Wu 2014). Furthermore, unusual mechanisms of protein folding have been speculated for some cellulases which may require assistance by specific chaperon(s) (Mingardon et al.2011). For instance, co-expression of chaperon-like B. subtilis PrsA protein was able to improve the secretion yield of heterologous amylase and penicillinase in Lc. lactis (Lindholm et al.2006). Increase of the secretion yield of heterologous cellulases may also be obtained by inactivation of housekeeping protease(s), as demonstrated by Lc. lactis mutants defective in the unique exported housekeeping protease HtrA (Wieczoreck and Martin 2010). Co-expression of protease inhibitors found as integral components of some clostridial cellulosomes (Meguro et al.2011; Xu et al.2014) could be an alternative strategy worth testing. Apart from improving the amount of the secretion yield of heterologous cellulases may also be obtained by inactivation of housekeeping protease(s), as demonstrated by Lc. lactis mutants defective in the unique exported housekeeping protease HtrA (Wieczoreck and Martin 2010). Co-expression of protease inhibitors found as integral components of some clostridial cellulosomes (Meguro et al.2011; Xu et al.2014) could be an alternative strategy worth testing. Apart from improving the amount of cellulolytic enzymes, future directions in the construction of recombinant cellulolytic LAB should focus on improving synergism of designer cellulase systems. Expression of multiple enzymes with highly complementary activities, preferably in a single strain, is essential for developing strains aimed at CBP of complex substrates. Apart from traditional cellulase activities (i.e. exoglucanases, endoglucanases and β-glucosidases, Fig. 3), attention should also be addressed to recently discovered cellulose-active proteins such as microbial expansins (Chen et al.2016) and lytic polysaccharide monooxygenases (LPMOs) (Liang et al.2014). The latter could significantly improve depolymerisation of most recalcitrant polysaccharides such as crystalline cellulose. Gene integration into the LAB chromosome appears to be the most suitable strategy for contructing genetically stable strains that co-express multiple cellulases. An extensive literature on integrative gene expression systems in LAB is available, although it is mainly focused on lactobacilli and Lc. lactis (for extensive reviews refer to Gaspar et al.2013 and Bravo and Landete 2017). Molecular tools for unlabelled (i.e. without insertion of antibiotic resistance markers) gene integration in the LAB genomic DNA include homologous recombination (e.g. pORI, pSEUDO and Cre-lox systems) or single-stranded DNA recombineering (Gaspar et al.2013; Bravo and Landete 2017). Some of them have already been used to improve pentose metabolism in different LAB strains (Table 3), but more extensive application to expression of heterologous hemi/cellulase systems seems necessary for significant progress of RCS in LAB. Surface display of proteins is also a valuable tool for increasing cellulase activity in LAB. This strategy mimics some of the most efficient cellulose depolymerisation systems found in nature (e.g. cellulosome), where cellulase activity is improved by rapid metabolism of cellulose hydrolysis products promoted by enzyme-cell proximity (Wieczoreck and Martin 2010). So far, studies in this direction have been reported by only two research groups, one led by Professor Martin in Canada (Wieczoreck and Martin 2010; 2012) and another coordinated by Professors Mizrahi and Bayer in Israel (Morais et al.2014; Stern et al.2018). While direct binding of glycosyl hydrolases to the LAB surface may cause allosteric hindrance and diminish enzyme/protein activity (Morais et al.2014; Stern et al.2018), surface display of mini-cellulosomes seems to be a good compromise for improving enzyme-cell synergism without major negative effects on cellulase flexibility and activity (Morais et al.2014). Furthermore, cellulosomes were shown to improve enzyme stability (Stern et al.2018). Multiple tools for protein surface-display in LAB through covalent (i.e. sortase-mediated) and non-covalent (e.g. LysM domains) binding have been reported (Okano et al.2008; Wieczoreck and Martin 2010; Morais et al.2014; Zadravec, Štrukelj and Berlec 2015) and can be used for further development of these strategies. CONCLUSION For a long time LAB have been used for industrial purposes and show good characteristics for future applications to second generation biorefinery. Generally, they can metabolise several monosaccharides which are components of plant biomass, including both hexoses and pentoses. Some of them can directly ferment short cello- or xylo-oligosaccharides or co-ferment hexoses and pentoses without carbon catabolite repression. Successful examples of LAB fermentation of hydrolysed lignocellulosic feedstocks (e.g. algal cake, corncob, corn stover, paper mill sludge, sugarcane bagasse, trimming vine shoots and wheat straw) have been reported. However, the high costs of physico-chemical pre-treatment and the large amounts of commercial cellulases needed for biomass saccharification are major barriers towards the industrial application of these technologies. While waiting for the development of cheaper pre-treatments or cellulase-production processes, research for alternative lignocellulose-LAB fermentation strategies is in progress. Synthetic consortia of cellulolytic microorganisms and LAB may eliminate the need for exogenous cellulases through an approach that mimics the natural microbial communities involved in plant biomass decay. The main challenge as represented here concerns maintaining such stable consortia at the industrial scale, but the studies reported in the literature encourage pursuing research along this rarely investigated path. Recombinant strategies aim to engineer LAB with heterologous cellulase systems which are able to directly ferment lignocellulose without any external help. This strategy promises huge cost reductions in processing, but is highly challenging. Despite the relatively high number of gene tools available for LAB, RCS suffer from the intrinsic toxicity of many heterologous cellulases and from lignocellulose recalcitrance requiring expression of multiple synergistic enzyme activities. Recombinant LAB obtained so far cannot grow on cellodextrins longer than 8–9 glucose units and intense research will be needed to produce direct fermentation of lignocellulosic feedstocks. In conclusion, useful progress towards LAB application in second generation biorefinery has been reported. Since finding alternative energies is currently a global priority, it is hoped that new resources will help support further developments in this research area. With this perspective, each of the strategies presented in this review represents a promising opportunity. Acknowledgements We would like to thank Professor Enrica Pessione for critical reading of the manuscript. 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TI - Alternative strategies for lignocellulose fermentation through lactic acid bacteria: the state of the art and perspectives
JF - FEMS Microbiology Letters
DO - 10.1093/femsle/fny126
DA - 2018-05-14
UR - https://www.deepdyve.com/lp/oxford-university-press/alternative-strategies-for-lignocellulose-fermentation-through-lactic-yl9uF0RZKk
SP - 1
VL - Advance Article
IS - 15
DP - DeepDyve
ER -