2019 Strain Engineering for Mi

Home , Glycerol dehydratase, Glycerol dehydrogenase, Methylglyoxal synthase, Propionate kinase

Biotechnology Advances 37 (2019) 538–568

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Biotechnology Advances

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Research review paper Strain engineering for microbial production of value-added chemicals and T fuels from glycerol ⁎ Adam W. Westbrook , Dragan Miscevic, Shane Kilpatrick, Mark R. Bruder, Murray Moo-Young, ⁎ C. Perry Chou

Department of Chemical Engineering, Waterloo, Ontario, Canada

ARTICLE INFO ABSTRACT

Keyword: While the widespread reliance on fossil fuels is driven by their low cost and relative abundance, this fossil-based Glycerol economy has been deemed unsustainable and, therefore, the adoption of sustainable and environmentally Metabolic engineering compatible energy sources is on the horizon. Biorefinery is an emerging approach that integrates metabolic Biorefinery engineering, synthetic biology, and systems biology principles for the development of whole-cell catalytic Biofuels platforms for biomanufacturing. Due to the high degree of reduction and low cost, glycerol, either refined or E. coli crude, has been recognized as an ideal feedstock for the production of value-added biologicals, though microbial Clostridium Klebsiella dissimilation of glycerol sometimes can be difficult particularly under anaerobic conditions. While strain de- Citrobacter velopment for glycerol biorefinery is widely reported in the literature, few, if any, commercialized bioprocesses Lactobacillus have been developed as a result, such that engineering of glycerol metabolism in microbial hosts remains an untapped opportunity in biomanufacturing. Here we review the recent progress made in engineering microbial hosts for the production of biofuels, diols, organic acids, biopolymers, and specialty chemicals from glycerol. We begin with a broad outline of the major pathways for fermentative and respiratory glycerol dissimilation and key end metabolites, and then focus our analysis on four key genera of bacteria known to naturally dissimilate glycerol, i.e. Klebsiella, Citrobacter, Clostridium, and Lactobacillus, in addition to Escherichia coli, and system- atically review the progress made toward engineering these microorganisms for glycerol biorefinery. We also identify the major biotechnological and bioprocessing advantages and disadvantages of each genus, and bottlenecks limiting the production of target metabolites from glycerol in engineered strains. Our analysis culmi- nates in the development of potential strategies to overcome the current technical limitations identified for commonly employed strains, with an outlook on the suitability of different hosts for the production of key metabolites and avenues for their future development into biomanufacturing platforms.

Abbreviations: 3-HPA, 3-hydroxypropionaldehyde; 1,2-PDO, 1,2-propanediol; 1,3-PDO, 1,3-propanediol; 1,3-PDOOR, 1,3-PDO oxidoreductase; 2,3-BDO, 2,3-butanediol; 3-HH, 3-hydroxyhexanoate; 3-HP, 3-hydroxypropionic acid; 3-HV, 3-hydroxyvalerate; (R)-3-HV-CoA, (R)-3-hydroxyvaleryl-CoA; ALDH, aldehyde dehydrogenase; ACE, Allele-Coupled Exchange; AOR, aldehyde oxidoreductase; asRNA, antisense RNA; ATP, adenosine Triphosphate; cAMP, cyclic adenosine monophosphate; CRE, catabolite repression element; CRISPR, Clustered Regularly Interspaced Palindromic Repeats; CRISPRi, CRISPR interference; Cas9, CRISPR- associated [protein] 9; CRP, cAMP receptor protein; dcw, dry cell weight; DHA, dihydroxyacetone; DHAK, DHA kinase; DHAP, dihydroxyacetone phosphate; DODHt, diol dehydratase; EDP, Entner-Doudoroff pathway; FAD, flavin adenine dinucleotide; FHL, formate hydrogen lyase; FDH, formate dehydrogenase; G3P, glycerol-3- phosphate; GDHt, glycerol dehydratase; GluDH, glucose dehydrogenase; GlyDH, glycerol dehydrogenase; GK, glycerol kinase; LDH, lactate dehydrogenase; MDH, malate dehydrogenase; MG, methylglyoxal; MGR, MG reductase; MGS, MG synthase; NADH, nicotinamide adenine dinucleotide; NOX, NADH oxidase; PHA, polyhydroxyalkanoate; P(3HB-co-3HH), poly(3-hydroxyburyrate-co-3-hydroxyhexanoate); P(3HB-co-3HV), poly(3-hydroxybutyrate-co-3-hydroxyvalerate); P(3HP), poly (3-hydroxypropionate); PDU, propanediol utilization; PEP, phosphoenolpyruvate; PCK, PEP carboxykinase; PFL, pyruvate formate lyase; PDC, pyruvate decarboxylase; PDH, pyruvate dehydrogenase; PFOR, pyruvate:ferrodoxin oxidoreductase; PGL, phosphogluconolactonase; PPC, PEP carboxylase; PPP, pentose phosphate pathway; PTA, phosphotransacetylase; PYK, pyruvate kinase; PO, pyruvate oxidase; RBS, ribosome binding site; Sbm, sleeping beauty mutase; TCA, tricarboxylic acid; UDH, pyridine nucleotide transhydrogenase ⁎ Corresponding authors. E-mail addresses: [email protected] (A.W. Westbrook), [email protected] (C.P. Chou). https://doi.org/10.1016/j.biotechadv.2018.10.006 Received 16 December 2017; Received in revised form 3 October 2018; Accepted 10 October 2018 Available online 17 October 2018 0734-9750/ © 2018 Elsevier Inc. All rights reserved. A.W. Westbrook et al. Biotechnology Advances 37 (2019) 538–568

1. Introduction only results in higher yields but also expands the repertoire of chemicals and fuels generated from microbial systems. However, microbial Currently, it is estimated that ~90% of the world’s energy re- dissimilation of glycerol is difficult under anaerobic conditions, asthe quirements are met through the use of non-renewable fossil fuels such cellular redox balance (i.e. the NAD+/NADH ratio) must be properly as petroleum, natural gas, bitumens, oil shale, and coal (Parikka, 2004; maintained through terminal transfer of electrons to internally pro- Pöschl et al., 2010; Srirangan et al., 2012). These resources, particularly duced organic compounds as opposed to molecular oxygen (Celińska, oil and natural gas, are also the most important feedstock for the pro- 2010; Yazdani and Gonzalez, 2007). As a result, only select organisms duction of fine and commodity chemicals (Basiago, 1994; Liu et al., (e.g. Clostridium pasteurianum and Klebsiella pneumoniae) are capable of 2010). Nevertheless, the unrestricted use of fossil fuels is considered fermenting glycerol anaerobically. Glycerol metabolism generates in- unsustainable due to the finite supply and unequal distribution of termediates such as dihydroxyacetone (DHA) and 3-hydro- natural reserves, coupled with climate change resulting from increasing xypropionaldehyde (3-HPA), and reduced end-products such as greenhouse gas emissions (Basiago, 1994; Börjesson, 2009; Srirangan ethanol, 1,2-propanediol (1,2-PDO), 1,3-propanediol (1,3-PDO), and et al., 2012). As a result, biorefinery becomes an emerging approach 2,3-butanediol (2,3-BDO) (Clomburg and Gonzalez, 2013; Murarka through integrating systems biology, genetic engineering, synthetic et al., 2008; Yazdani and Gonzalez, 2007). Accordingly, glycerol fer- biology, and metabolic engineering principles for the development of mentation has been extensively explored in both native and genetically whole-cell biocatalytic platforms for manufacturing purposes (Menon tractable hosts for the production of various value-added chemicals and and Rao, 2012; Octave and Thomas, 2009). While this appears to be a fuels, including advanced alcohols (e.g. 1-propanol), organic acids (e.g. promising avenue, a major detriment to the use of biological platforms succinic, propionic, and 3-hydroxypropionic acids), natural products is the high cost of feedstock (Menon and Rao, 2012; Octave and (e.g. terepenes), ketones (e.g. acetone and butanone) and bio(co)poly- Thomas, 2009). Industrially, value-added products are derived from mers (e.g. polyhydroxyalkanoate (PHA) and poly(3-hydroxybutyrate- agricultural crops (i.e. first-generation feedstock) or from lig- co-3-hydroxyvalerate) [P(3HB-co-3HV)]). This article reviews our cur- nocellulosic crops and agricultural wastes (i.e. second-generation rent understanding of glycerol metabolism and recent approaches of feedstock) (Srirangan et al., 2012). First-generation feedstocks (e.g. strain engineering for glycerol biorefinery. corn, starch, oilseed, and sugar) often have a high energy, oil, and carbohydrate content and are currently used for the production of 2. Overview of glycerol metabolism in microbes biodiesel (and other bio-esters), bioethanol (and other bioalcohols), and biogas (Hein and Leemans, 2012; Srirangan et al., 2012). However, Due to the high reductance of glycerol, there is an excess of reducing these bioprocess schemes are considered unsustainable due to their equivalents that remain unbalanced such that the overall redox balance competition with the human food and animal feed markets, and the within a cell is a vital principle that governs the nature and quantity of requirement for large arable lands (Schmidhuber, 2008). While not metabolites during glycerol dissimilation (Clomburg and Gonzalez, directly impacting the cost and availability of food supplies, second- 2013). The natural fermentative metabolism of glycerol has been generation feedstocks are not practical due to their inherent recalci- thoroughly studied in the Enterobacteriaceae family (Booth, 2005; trance and the high cost of pre-treatment technologies required for their Murarka et al., 2008). Under fermentative conditions, microorganisms valorization (Luo et al., 2010; Srirangan et al., 2012). must be metabolically capable of consuming glycerol in the absence of Accordingly, several alternative biorefinery schemes have been external electron acceptors. Specifically, for 1.1 mM of glycerol in- proposed to utilize biomass feedstock in an economically viable manner corporated into cell mass, 0.6 mM reducing equivalents (i.e. NADH) are to compete with existing petroleum-refinery technologies. One such generated. This is not an issue for other common carbohydrates, such as model is co-utilization of the starting feedstock and the byproduct glucose and xylose, since their degree of reduction is lower than that of stream (Clomburg and Gonzalez, 2013; Yazdani and Gonzalez, 2007). the cell biomass. Furthermore, excess electrons cannot be consumed For instance, glycerol is a major sugar alcohol byproduct associated through the production of reduced products such as ethanol or succi- with bioethanol (Raynaud, and xe, line, Sar, xe, abal, P., Meynial-Salles, nate since their production from glycerol is already redox-balanced. I., Croux, C., Soucaille, P., 2003; Zeng and Biebl, 2002) and biodiesel Thus, to achieve redox poise, there must be a metabolic pathway gen- (Balat and Balat, 2010) production. Transesterification of oils and fats erating more reduced products for consuming surplus reducing with an alcohol generates ~1 lb crude glycerol (with at least 80% equivalents. The formation of 1,3-PDO (Homann et al., 1990; Saint- purity) for every 10 lb biodiesel produced (López et al., 2009; Sabourin- Amans et al., 1994; Schütz and Radler, 1984) and 1,2-PDO (Clomburg Provost and Hallenbeck, 2009). Likewise, all first-generation-based and Gonzalez, 2011) via ATP-neutral pathways results in successful bioethanol programs generate significant amounts of glycerol as a fer- anaerobic dissimilation of glycerol due to the highly reduced nature of mentative end-product (Kim et al., 2008; Zeng and Biebl, 2002). Cur- these metabolites (κ = 5.33). rently, biodiesel and bioethanol represent the two largest biomass en- Like other small and uncharged molecules, glycerol can cross the ergy programs in the world, and their significant growth led to a more cytoplasmic membrane via two mechanisms: (i) passive diffusion under than 10-fold increase in global glycerol production between 2004 and high concentrations; and (ii) facilitated diffusion with the aid ofa 2011, with ~700 million lb crude glycerol produced in the United transport protein (i.e. GlpF) under low concentrations (da Silva et al., States alone in 2011 (Clomburg and Gonzalez, 2013; López et al., 2009; Sun et al., 2008). Intracellular glycerol is subsequently metabo- 2009). Additionally, many other industries which derive products from lized either in the presence of electron acceptors (i.e. respiratory animal fats and vegetable oils generate waste streams of high glycerol branch) or in the absence of electrons acceptors (i.e. fermentative content, e.g. the oleochemical industry (Sabourin-Provost and branch) (Gonzalez et al., 2008). Fermentative metabolism of glycerol Hallenbeck, 2009; Yazdani and Gonzalez, 2007). This surplus in gly- has been previously reported in microbes within the genera of Klebsiella cerol has plummeted its price to ~2.5 cents/lb in recent years, effec- (Forage and Foster, 1982; Homann et al., 1990), Citrobacter (Daniel tively rendering it a waste product with an associated disposal cost (Raj et al., 1995; Seifert et al., 2001), Clostridium (Abbad-Andaloussi et al., et al., 2008). 1995; Biebl, 2001; Macis et al., 1998), and Lactobacillus (Talarico et al., As a carbon source, glycerol offers several distinctive advantages 1990), as well as in Escherichia coli (Dharmadi et al., 2006; Murarka over traditional fermentable sugars. Due to the high degree of reduction et al., 2008). The natural fermentative bioconversion of glycerol to a (reductance κ = 4.67), glycolytic degradation of glycerol generates more reduced metabolite, such as 1,3-PDO, occurs both reductively and approximately twice the number of reducing equivalents (i.e. NADH) oxidatively in Klebsiella sp., Citrobacter sp., Clostridium sp., and certain compared to xylose and glucose (κ = 4) (Murarka et al., 2008; Yazdani strains of Lactobacilli sp. (da Cunha and Foster, 1992; da Silva et al., and Gonzalez, 2007). Therefore, harnessing glycerol metabolism not 2009). In the reductive branch, a B12-dependent glycerol dehydratase

539 A.W. Westbrook et al. Biotechnology Advances 37 (2019) 538–568

Fig. 1. Schematic representation of the major glycerol catabolism pathways in bacteria. Blue arrows represent reactions in the fermentative branch, displaying both reductive and oxidative routes; green arrows represent reactions in the respiratory branch; red arrows represent reactions in the 1,2-PDO pathway; and gold arrows represent reactions in the 2,3-BDO pathway. Solid line: direct bioconversion step; dashed line: multi-step bioconversion. Enzyme abbreviations: ACK, acetate kinase; ADH, bifunctional aldehyde/alcohol dehydrogenase; ae-G3PDH, aerobic glycerol-3-phosphate dehydrogenase; AKR, aldo-keto reductase; ALDH, aldehyde dehydrogenase; ALDC, α-acetolactate decarboxylase; ALS, α-acetolactate synthase; an-G3PDH, anaerobic glycerol-3-phosphate dehydrogenase; AR, acetoin reductase; DODHt, diol dehydratase; DHAK, dihydroxyacetone kinase; FHL, formate hydrogen lyase; FRD, fumarate reductase; GDHt, glycerol dehydratase; GK, glycerol kinase; GlpF, glycerol diffusion facilitator; GlyDH, glycerol dehydrogenase; LDH, lactate dehydrogenase; MGR, methylglyoxal reductase; MGS, methylglyoxal synthase; PDH, pyruvate dehydrogenase; PduL, phosphate propanoyltransferase; PduP, propionaldehyde dehydrogenase; PduW, propionate kinase; PFL, pyruvate formate-lyase; PTA, phosphotransacetylase; PK, pyruvate kinase; 1,2-PDOOR; 1,2-propanediol oxidoreductase; 1,3-PDOOR, 1,3-propanediol oxidoreductase. Chemical intermediates and product abbreviations: α-AL, α-acetolactate; acetyl-P, acetyl phosphate; DHA, dihydroxyacetone; DHAP, dihydroxyacetone phosphate; G3P, glycerol-3- phosphate; MG, methylglyoxal; PEP, phosphoenolpyruvate; 1,2-PDO, 1,2-propanediol; 1,3-PDO, 1,3-propanediol; 3-HP, 3-hydroxypropionate; 3-HP-CoA, 3-hydroxypropionyl-CoA; 3-HP-P, 3-hydroxypropionyl phosphate.

(GDHt) dehydrates glycerol to form 3-HPA, which is subsequently re- to glycerol metabolism (i.e. DhaB/DhaBCE, DhaT, DhaD, and DhaK) are duced to 1,3-PDO (Fig. 1)(Murarka et al., 2008). In the latter reaction, located in a DHA (dha) regulon (Zhu et al., 2002). In certain Clostridium NAD+ is regenerated by NADH-dependent 1,3-PDO oxidoreductase sp., such as Clostridium butyricum, the dha regulon is composed of three (1,3-PDOOR), resulting in a balanced internal redox state (González- genes encoding GDHt (i.e. DhaB1 encoded by dhaB1), its reactivating Pajuelo et al., 2006). Glycerol fermentation is the only known process protein (i.e. DhaB2 encoded by dhaB2), and 1,3-PDOOR (i.e. DhaT for anaerobic conversion for 1,3-PDO production (Homann et al., encoded by dhaT)(Raynaud, and xe, line, Sar, xe, abal, P., Meynial- 1990). As 1,3-PDO is more reduced than glycerol, its formation is ac- Salles, I., Croux, C., Soucaille, P., 2003), while the dha regulon can be companied by a more oxidized co-product (Clomburg and Gonzalez, found in two spatially different clusters on the chromosome of Clos- 2013). In Klebsiella sp. and Citrobacter sp., acetic acid is the major co- tridium perfringens (Ignatova et al., 2003)(Table 1). product during 1,3-PDO production under fermentative conditions E. coli can ferment glycerol anaerobically in a pH-dependent manner (Homann et al., 1990), whereas mixed co-products are formed (i.e. without external electron acceptors (Booth, 2005; Dharmadi et al., butyrate, butanol, ethanol, and lactate) in Clostrodium sp. (Biebl, 2001; 2006). The internal availability of CO2 generated by formate hydrogen Clomburg and Gonzalez, 2013; Saint-Amans et al., 2001). Biosynthesis lyase (FHL)-mediated oxidation of formate enables E. coli to anaerobi- of 1,3-PDO relies on the availability of NADH, which can be naturally cally metabolize glycerol. For E. coli to synthesize small molecules and generated in Klebsiella sp. and Lactobacillus sp. via oxidation of 3-HPA fatty acids, it requires a steady supply of bicarbonate/CO2 substrates. into 3-hydroxypropionic acid (3-HP) by NAD+-dependent aldehyde Lowering the pH of the media as well as potassium and phosphate dehydrogenase (ALDH) (Zhu et al., 2015). In the oxidative branch, concentrations under fermentative conditions activates the transcrip- + NAD -dependent glycerol dehydrogenase (GlyDH) catalyzes the tion of FHL and subsequently achieves the cellular requirement for CO2 transformation of glycerol to dihydroxyacetone (DHA) (Fig. 1), which is upon formate accumulation (Murarka et al., 2008). Additionally, for- subsequently phosphorylated by a DHA kinase (DHAK) to form dihy- mate oxidation also produces H2 and, therefore, can contribute to re- droxyacetone phosphate (DHAP) (Shams Yazdani and Gonzalez, 2008). ducing equivalent accumulation that other redox-balanced pathways, In Klebsiella sp. and Citrobacter sp., the genes encoding enzymes specific such as succinate and ethanol-generating pathways, cannot (Dharmadi

540 ..Wsboke al. et Westbrook A.W. Table 1 Summary of enzymes comprising natural pathways for glycerol catabolism in Klebsiella sp., Citrobacter sp., Clostridium sp., Lactobacillus sp., and E. coli. Locus tags, and respective enzyme names where available, are provided for a representative strain for each genus or species. Each numbered column corresponds to one of the numbered reactions in Fig. 1.

Fermentative metabolism Respiratory metabolism

Organism 1 2 3 4 5 6 7 8 9 ⁎ ⁎ Klebsiella sp.1,2 GDHt: 1,3-PDOOR: ALDH: PduL PduW GlyDH: DHAK: GK: ⁎ ⁎ ⁎ ⁎ ⁎ ⁎ ⁎ (K. pneumoniae DhaB , a DhaT YdcW PduP (RS17225) (RS17275) DhaD DhaKI GlpK , d MGH 78578) (RS18900, (RS18910), (RS10355), (RS17245) (RS18930), (RS18955), (RS21635) ⁎ ⁎ ⁎ ⁎ RS18895, YqhD , b YneI , c GldA DhaKII RS18890) (RS18380) (RS08785), (RS22860) (RS18940, ⁎ FeaB RS18945, DODHt: (RS07805), RS18950) ⁎ PduCDE PuuC ⁎ (RS17190 , (RS05455), ⁎ RS17195, BadH RS17200) (RS03160), ⁎ GabD (RS01360) Citrobacter 1,3-PDODH: NA NA NA NA GlyDH: GK: ⁎ ⁎ sp.3,4 GDHt: DhaT ,b DhaD ,c DHAK: GlpKd ⁎ ⁎ (C. freundii DhaBCE (RS02580) (RS02600) DhaK (RS05005) CFNIH1) (RS02570, GldA (RS19390) RS02565, (RS07260) RS02560)

DODHt: 541 PduCDE (RS20865, RS20870, RS20875) Clostridium GDHt: 1,3-PDOH: NA NA NA NA GlyDH: DHAK: GK: sp.5,6 PduCDE/DhaBCE DhaT2 GldA DhaK GlpK (C. pasteurianum (RS11085, (RS11050) (RS05875, (RS20060) (CA_C1321)k ATCC 6013) RS11080, DhaT1 RS18665), RS11075) (RS05350) RS13630, ⁎ DhaB1 RS16340 (Genbank AY112989.1)j ⁎ Lactobacillus DODHt: 1,3-PDOOR: ALDH: PduP PduL PduW GlyDH: NA GK: ⁎ ⁎ ⁎ sp.7,8 PduCDE RS00160 , PduP ,e (RS09045) (RS09070) (RS09035) RS09575 RS05600 ⁎ (L. reuteri (RS09105, RS09040 (RS09045) DSM 20016) RS09100,

RS09095) Biotechnology Advances37(2019)538–568 E. coli NA ALDR: ALDH: NA NA NA GlyDH: DHAK: GK: ⁎ ⁎ ⁎ ⁎ ⁎ YqhD AldH ,c GldA DhaKLM GlpK (b3011) (b1300) (b3945) (b1200, (b3926) b1199, b1198)

Respiratory metabolism 1,2-PDO Succinate Lactate 2,3-BDO

Organism 10 11 12 13 14 15 16 17 18 19 20 Klebsiella sp.1,2 ae-G3PDH: an-G3PDH: NA NA NA NA NA FRD: PK: LDH: ALS: ⁎ ⁎ (K. pneumoniae GlpD GlpABC FrdABCD PykA LdhA BudB MGH 78578) (RS20485) (RS14215, (RS24550, (RS12780), (RS07775), (RS11100) (continued on next page) ..Wsboke al. et Westbrook A.W. Table 1 (continued)

Respiratory metabolism 1,2-PDO Succinate Lactate 2,3-BDO

RS14220, RS24545, PykF Ldh1 RS14225) RS24540, (RS11500) (RS08770), RS24535) Ldh2 (RS21315) Citrobacter ae-G3PDH: an-G3PDH: NA NA NA NA PK: LDH: NA ⁎ sp.3,4 GlpD GlpABC FRD: RS16640, LdhA (C. freundii (RS04715) (RS22435, FrdABCD RS20115 (RS18135) CFNIH1) RS22440, NA (RS08335, RS22445) RS08330, RS08325, RS08320) Clostridium NA an-G3PDH: NA NA NA NA NA NA PK: LDH: NA sp.5,6 GlpA RS13430 RS13420, (C. pasteurianum (CA_C1322)k RS08505 ATCC 6013) Lactobacillus ae-G3PDH: NA NA NA NA NA NA FRD: PK: LDHf: NA sp.7,8 RS01940 RS08040 RS04025 RS00995, (L. reuteri RS03840, DSM 20016) RS03845, RS08830, RS09895 E. coli ae-G3PDH: an-G3PDH MGS: AKR: glyDH: MGR 1,2-PDOR: FRD: PK: LDH: NA ⁎ ⁎ ⁎ ⁎ ⁎ ⁎ ⁎ ⁎ ⁎ GlpD GlpABC MgsA YeaE GldA YdjG FucO FrdABCD PykA LdhA (b1380) (b3426) (b2241, b2242, (b0963) (b1781), (b3945) (b1771) (b2799) (b4154, (b1854), ⁎ ⁎ b2243) YghZ b4153, PykF 542 (b3001), b4152, (b1676) ⁎ YafB b4151) (b0207), ⁎ YqhE (b3012), ⁎ YqhD (b3011)

2,3-BDO Ethanol Acetate

Organism 21 22 23 24 25 26 27 28 29 Klebsiella sp.1,2 AR: ALDC: AR: PFL: PDC: ALDH: ADH: PTA: ACK: ⁎ ⁎ ⁎ ⁎ ⁎ (K. pneumoniae BudC BudA BudC PflAB RS14705 AldA YigB Pta AckA MGH 78578) (RS11105) (RS11095) (RS11105) (RS05010, PDH: (RS08050) (RS25235), (RS14425), (RS14420) RS05015) AceE RS02890, RS22680, EutD TdcD (RS00635), RS05425, YqhDb (RS14985) (RS12355) ⁎ PoxB AstD (RS18380), Biotechnology Advances37(2019)538–568 ⁎ (RS04870) (RS06540) AdhE DHLAT: (RS11855), AceF AdhP (RS00640) (RS09985) Citrobacter NA NA NA PFL: PDH: ALDH: PTA: ACK: sp.3,4 PflA AceEF RS16255, ADH: Pta RS20950, (C. freundii (RS14340) (RS10115, RS20920 RS02285, (RS22670) RS22665 CFNIH1) RS10120) AstD RS04980, TdcD (RS16255) RS15860, (RS02795) RS16775, RS17775 Clostridium NA NA NA NA ALDH: ADH: ACK: sp.5,6 AdhE AdhE AckA (continued on next page) ..Wsboke al. et Westbrook A.W. Table 1 (continued)

2,3-BDO Ethanol Acetate

(C. pasteurianum PDH: (RS07340, (RS07340, PTA: (CLPA_ ATCC 6013) NifJ RS02750, RS02750, Pta RS10110) (RS02460) RS07580 RS07580 (RS10115) RS14065, RS14065, CA_P0162k, CA_P0162k, CA_P0162k) CA_P0035k) Lactobacillus NA NA NA NA PDH: ALDH: ADHg: PTA: ACK: sp.7,8 RS03410, RS00180, RS07840, RS02075 RS02910, (L. reuteri RS03415 RS01680 RS08065, RS09035e DSM 20016) DHLAT: RS08320, RS03420 RS09040, RS09680 E. coli NA NA NA PFL: PDH (E1): ALDHh: ADHi: PTA: ACK: ⁎ ⁎ ⁎ ⁎ ⁎ ⁎ PflB AceE AdhE (b1241) AdhE (b1241), Pta AckA ⁎ (b0903), (b0114) AdhP (b1478) (b2297), (b2296) ⁎ ⁎ TdcE DHLAT (E2): EutD (b2458) ⁎ (b3114) AceF (b0115) LADH (E3): ⁎ Lpd (b0116)

1K. pneumoniae and K. oxytoca; 2 locus tag prefix is KPN; 3 4 locus tag prefix is CFNIH; 5C. pasteurianum and C. acetobutylicum; 6 locus tag prefix is CLPA; 7L. reuteri, L. panis, L. collinoides, L. diolivorans, and L. brevis; 8 locus tag prefix is LREU; a inactive in K. oxytoca; b NAD(P)H-dependent; c NAD(P)+-dependent; d Potentially inactive pathway in some strains; e homologue not present in L. panis; f identified putative LDHs were selected based g

543 on the most significant homology among putative LDHs found in all species included (additional putative LDHs may exist in eachspecies); identified putative ADHs were selected based on the most significant homology h i j k among putative ADHs found in all species included (additional putative ADHs may exist in each species); additional ADHE may exist; additional ADH may exist; B12 -independent GDHt from C. butyricum; from C. ⁎ acetobutylicum ATCC 824; function of enzyme experimentally demonstrated. Biotechnology Advances37(2019)538–568 A.W. Westbrook et al. Biotechnology Advances 37 (2019) 538–568 et al., 2006). Also, under fermentative conditions, E. coli can recycle the Wang et al., 2017b). Components of the oxidative and reductive bran- excess H2 formed via FHL by functional expression of native hydro- ches of the fermentative pathway of glycerol dissimilation (i.e. DhaD, genase isoenzymes Hyd-1 and Hyd-2 (Murarka et al., 2008; Sawers DhaKII, DhaB, and DhaT) were expressed under aerobic conditions in a et al., 1985; Sawers and Boxer, 1986). Compared to 1,3-PDO, the K. pneumoniae strain lacking a functional glp regulon, although the conversion of glycerol to 1,2-PDO is uncommon and less effective with expression levels were markedly lower compared to those observed significantly lower yields (Clomburg and Gonzalez, 2013; Zeng and under anaerobic conditions (Forage and Lin, 1982). Conversely, gly- Sabra, 2011). In the oxidative branch, 1,2-PDO production starts with cerol kinase (i.e. GlpK encoded by glpK) and G3P dehydrogenase (GlpD the formation of DHAP via phosphorylation of DHA. Subsequently, encoded by glpD) were expressed under aerobic conditions in a strain DHAP enters the methylglyoxal (MG) pathway and is converted to the possessing a functional glp regulon, while the expression of DhaD, intermediate MG by a MG synthase (MGS; Fig. 1)(Hopper and Cooper, DhaKII, DhaB, and DhaT were highly repressed. Note that 1,3-PDO 1972). The availability of DHAP is crucial since it represents a node production under microaerobic conditions has been studied extensively where carbon flux between glycolysis and 1,2-PDO formation diverges (Chen et al., 2003a, 2003b; Chen et al., 2003a, 2003b; Liu et al., 2007; (Clomburg and Gonzalez, 2011). Conversion of MG to 1,2-PDO is pos- Mu et al., 2006; Xiu et al., 2007; Zhang and Xiu, 2009). Interestingly, sible through either (i) the intermediate acetol via aldehyde oxidor- the addition of exogenous cyclic adenosine monophosphate (cAMP), eductase (AOR), or (ii) the intermediate lactaldehyde via GlyDH or MG the inducer of the cAMP receptor protein (CRP) that relieves catabolite reductase (MGR; Fig. 1)(Clomburg and Gonzalez, 2011). repression in the absence of preferred carbon sources (Zheng et al., Under respiratory conditions, glycerol dissimilation can advance 2004), significantly increased the expression of DhaD, DhaKII, DhaB, through two different two-step routes to DHAP (Fig. 1): (i) the aerobic and DhaT under aerobic conditions in either strain, although the fold glycerol kinase (GlpK)-glycerol-3-phosphate (G3P) dehydrogenase increase in expression was significantly higher in the strain possessing a (GlpD) pathway; or (ii) the anaerobic GlpK-anaerobic G3P dehy- functional glp regulon (Forage and Lin, 1982). As the minor oxidized drogenase (GlpABC) pathway (Blankschien et al., 2010; Durnin et al., product derived from the reductive branch of fermentative glycerol 2009). In either case, G3P is first synthesized via phosphorylation of dissimilation, 3-HP is derived from 3-HPA by the NAD+-dependent glycerol by GlpK at the expense of ATP (Fig. 1)(Murarka et al., 2008), (YdcW encoded by ydcW) and NADP+-dependent (YneI encoded by or it can be directly imported into the cell via the G3P transporter, GlpT yneI) ALDHs (Fig. 1)(Luo et al., 2013; Luo et al., 2011a, 2011b). 3-HP (encoded by glpT)(Jin et al., 1982). Through the aerobic pathway, production can provide compensatory NADH for 1,3-PDO synthesis GlpD, a plasma membrane-associated homodimer, generates DHAP via (Zhu et al., 2015), and alleviate the accumulation of the toxic inter- oxidation of G3P. On the other hand, DHAP is synthesized from G3P by mediate 3-HPA, which is also known to inhibit DhaT in the presence of GlpABC (encoded by glpABC) anaerobically (Fig. 1). In addition to excess glycerol (Hao et al., 2008a; Sun et al., 2008). Moreover, the being a precursor to DHAP, G3P also serves as a precursor for lipid expression of other ALDHs, including PuuC (Ashok et al., 2013a; Ashok biosynthesis or production of other metabolites (da Silva et al., 2009). et al., 2011; Huang et al., 2012; Luo et al., 2011a, 2011b), FeaB (Huang Similar to the dha regulon in the fermentative pathway, the glp regulon et al., 2012), BadH (Huang et al., 2012), and GabD (Huang et al., 2012) in E. coli contains genes encoding enzymes found in the respiratory (encoded by puuC, feaB, badH, and gabD, respectively), can potentially pathway (i.e. GlpF, GlpK, GlpD, and GlpABC). Similar glp regulons are enhance 3-HP production in K. pneumoniae. Finally, alternate pathways also present in Klebsiella sp. and Citrobacter sp. (da Silva et al., 2009), derived from the propanediol utilization (PDU) system exist for the while the putative partial glp regulon of Lactobacillus sp. is inactive, due, conversion of glycerol to 1,3-PDO and 3-HP in Klebsiella sp. (Bobik in part, to the absence of DHAK (Morita et al., 2008). et al., 1999; Cho et al., 2015; Luo et al., 2012), as is the case in Sal- monella sp. (Bobik et al., 1999) and Lactobacillus sp. (Makarova et al., 3. Biological conversion of glycerol for the production of value- 2006; Morita et al., 2008; Sauvageot et al., 2002; Sriramulu et al., added chemicals in select microbial platforms 2008). In this system, glycerol is first converted to 3-HPA by12 theB - dependent diol dehydratase (DODHt) PduCDE (encoded by pduCDE) 3.1. Klebsiella sp. (Bobik et al., 1999; Cho et al., 2015; Morita et al., 2008; Sauvageot et al., 2002) with the reactivation factor PduGH (encoded by pduGH) 3.1.1. Natural metabolism (Bobik et al., 1999), followed by conversion of 3-HPA to 1,3-PDO via a Klebsiella sp. (i.e. K. pneumoniae) own natural pathways for flexible 1,3-PDOOR (e.g. DhaT). Alternatively, 3-HPA can be converted to 3-HP- and efficient metabolism of glycerol (Kumar and Park, 2017; Zeng CoA via PduP, a CoA-dependent propionaldehyde dehydrogenase en- et al., 1993; Zhang et al., 2008; Zhang et al., 2009). Fermentative coded by pduP, followed by phosphorylation of 3-HP-CoA to yield 3-HP- glycerol dissimilation in Klebsiella sp. proceeds anaerobically through P via a phosphate propanoyltransferase PduL (encoded by pduL), and coupled oxidative and reductive branches (Fig. 1), providing outlets for final conversion of 3-HP-P to 3-HP via a propionate kinase PduW (en- energy generation and redox balance. In the oxidative branch, glycerol coded by pduW; Fig. 1)(Bobik et al., 1999; Honjo et al., 2015; Luo et al., is first converted to DHA by GlyDH (i.e. DhaD encoded by dhaD), and 2012). DHA is subsequently converted to DHAP by one of two DHAKs, i.e. During anaerobic growth, pyruvate metabolism is mediated by both DhaK I (encoded by dhaK) and DhaK II (encoded by dhaK123), with the pyruvate formate lyase (PFL) and pyruvate dehydrogenase (PDH) DhaK II playing the dominant role (Wang et al., 2003; Wei et al., 2014). complexes, with both enzyme systems presenting similar activities, Glycerol is simultaneously converted to 3-HPA by GDHt (i.e. DhaB while pyruvate:ferrodoxin oxidoreductase (PFOR) does not appear to be encoded by dhaB123), followed by reduction of 3-HPA to 1,3-PDO, active (Menzel et al., 1997) except under conditions of glycerol excess which is the major redox valve for glycerol dissimilation, via the NADH- (Zeng et al., 1993). The activity of PFL was observed to increase and dependent (DhaT encoded by dhaT) or putative NADPH-dependent decrease under glycerol limitation and excess, respectively (Menzel (YqhD encoded by yqhD) 1,3-PDOORs (Ashok et al., 2011; Wang et al., et al., 1997). Conversely, the activity of the PDH complex increases

2003; Zeng et al., 1993). In Klebsiella sp., which synthesize B12 natu- with glycerol concentration (Menzel et al., 1997). The phenomena of rally, DhaB is B12-dependent and is activated by GdrAB (encoded by oscillation of biomass concentration and glycerol consumption in con- gdrAB), a reactivation factor that can reactivate glycerol- or oxygen- tinuous anaerobic cultures of K. pneumoniae has been, in part, attrib- deactivated DhaB and protect it from substrate inhibition (Sun et al., uted to the feedback control between the parallel pyruvate metabolism

2003). Biosynthesis of 1,3-PDO can be limited by dhaB123 expression pathways for the production of acetate, CO2, 2,3-BDO, lactate, and (Ahrens et al., 1998; Wang et al., 2003) given the detrimental effects of ethanol (Menzel et al., 1996). Interestingly, the activity of PFL, which is 3-HPA accumulation (Wang et al., 2003). While a glp regulon is present the presumptive major player in fermentative metabolism of pyruvate, in K. pneumoniae, it is dormant in certain strains (Forage and Lin, 1982; is strongly affected by oscillations in anaerobic continuous cultures,

544 A.W. Westbrook et al. Biotechnology Advances 37 (2019) 538–568 while the activities of pyruvate kinase (PYK) and the PDH complex PDO production. On the other hand, co-fermentation of glycerol and were marginally influenced (Menzel et al., 1998). Finally pyruvate glucose is an alternate strategy to enhance 1,3-PDO production in the oxidase (PO encoded by poxB) appears to play a significant role in both Δdha background, and can be achieved via mutation of crr, encoding aerobic and anaerobic pyruvate metabolism, as mutation of poxB re- glucose phosphotransferase protein (EIIAGlc)(Oh et al., 2013a; Wang sulted in excessive acetate accumulation and severely inhibited cell et al., 2017c). Co-feeding limiting amounts of glucose with excess growth during aerobic cultivation, and abolished CO2 production under glycerol may stimulate biomass accumulation and NADH regeneration anaerobic conditions (Lin et al., 2016). via glucose metabolism, while glycerol can be converted to 1,3-PDO in Other major fermentative products produced by Klebsiella sp. in- the absence of oxidative glycerol metabolism. clude 2,3-BDO, acetate, lactate, succinate, and ethanol (Xu et al., 2009; The expression of genes in the reductive branch of the fermentative Zeng et al., 1996). A substantial amount of NADH released through the glycerol dissimilation pathway can increase carbon flux toward 1,3- activities of DhaD and GlpD is oxidized and discharged as H2 through PDO production with reduced 3-HPA accumulation, while expression of pathways other than the 1,3-PDO, ethanol, lactate, and 2,3-BDO path- genes in the oxidative branch can improve glycerol dissimilation and ways. When glycerol is in excess, the bulk of reducing equivalents are NADH levels albeit at the expense of potential side metabolite forma- consumed for 1,3-PDO production with a small amount being released tion. To alleviate growth cessation associated with toxic 3-HPA, native as H2. Under glycerol limitation, ethanol production is more prevalent dhaT was overexpressed (Hao et al., 2008b). While cell growth con- as it is energetically favorable, although ethanol is more toxic to Kleb- tinued until glycerol was exhausted with reduced levels of all major side siella sp. than 1,3-PDO (Zeng et al., 1993). metabolites, 1,3-PDO production was not improved (Hao et al., 2008b), even in fedbatch cultures where a decrease in the growth rate was 3.1.2. Strain engineering observed, eventually leading to growth stagnation (Zhao et al., 2009). Table 2 summarizes various strategies for strain engineering of Similarly, overexpression of native dhaD reduced ethanol and 2,3-BDO Klebsiella sp. to convert glycerol to value-added products, while detailed accumulation without improving 1,3-PDO formation, suggesting that technical discussion is provided in the following sections. the redox balance in the cell is optimal for natural 1,3-PDO synthesis (Zhao et al., 2009). On the other hand, coexpression of native dhaD and 3.1.2.1. 1,3-PDO. To date, 1,3-PDO has been the most common target dhaT significantly reduced 3-HPA accumulation, leading to asub- metabolite in engineered Klebsiella sp. The formation of 1,3-PDO stantial increase in 1,3-PDO production (Chen et al., 2009). simultaneously generates 1 mol NADH per mol glycerol consumed as Coexpression of a putative 1,3-PDOOR bearing similarity to YqhD the major redox valve for oxidative glycerol metabolism via the from E. coli and native gdrAB significantly increased the 1,3-PDO titer, glycolytic pathway. Major strain engineering strategies to enhance relative to coexpression of native dhaT and gdrAB, in a strain with the 1,3-PDO formation include: (1) disruption of the oxidative branch of oxidative branch of fermentative glycerol metabolism being disrupted the fermentative glycerol dissimilation pathway; (2) overexpression of (Seo et al., 2010). The activity of YqhD is NADPH-dependent, and, in genes in the reductive and/or oxidative branch of the fermentative contrast to DhaT, the overexpression of the putative 1,3-PDOOR could glycerol dissimilation pathway; and (3) inactivation of pathways potentially mitigate the NAD+/NADH imbalance resulting from dis- leading to side metabolites, such as lactate, 2,3-BDO, ethanol, ruption of the oxidative branch of fermentative glycerol metabolism. succinate, and acetate. Note that these strategies have been applied to However, glycerol consumption and 1,3-PDO production rates were balance the NAD+/NADH ratio, increase carbon flux toward 1,3-PDO, significantly lower relative to the parent strain coexpressing yqhD and and/or reduce toxic metabolite formation. The oxidative branch of gdrAB, in which the oxidative branch was intact, although the final 1,3- fermentative glycerol dissimilation produces DHAP, which is PDO titer was comparable (Seo et al., 2010). In addition, coexpression subsequently converted to the glycolytic intermediate glyceraldehyde of native dhaT and yqhD from E. coli reduced side metabolite formation, 3-phosphate. Glycolysis results in the synthesis of phosphoenolpyruvate but provided only a marginal increase in the 1,3-PDO titer (Zhuge et al., (PEP), leading to the formation of succinate or pyruvate, the latter of 2010), while yqhD expression alone reduced the rate of glycerol dis- which is an intermediate in 2,3-BDO, lactate, acetate, and ethanol similation without affecting the 1,3-PDO titer (Oh et al., 2013b). Ex- formation (Fig. 1). To inactivate oxidative glycerol metabolism in K. pression of yqhD facilitated high 1,3-PDO titers in E. coli (Emptage pneumoniae, dhaD and dhaK were mutated with the simultaneous use of et al., 2003a), although it seems to play a minor role in reductive gly- the lacZ promoter from E. coli to regulate the expression of dhaB123, or cerol metabolism in Klebsiella sp., and disruption of native dhaT nearly dhaR, encoding the transcription factor that activates the expression of abolishes 1,3-PDO production (Seo et al., 2009). Finally, basal expres- the dha regulon (DhaR), was mutated (Seo et al., 2009). In either sion of native puuC from the leaky tac promoter increased and de- approach, the reductive branch of fermentative glycerol dissimilation creased 1,3-PDO titers in cultures of lactate and lactate/2,3-BDO defi- was also inactivated, necessitating the expression of native dhaT and cient mutants, respectively (Zhu et al., 2015). The conversion of 3-HPA gdrAB to restore 1,3-PDO production. Side metabolite formation was to 3-HP (via PuuC) reduces 3-HPA levels, while producing NADH re- markedly reduced, although 1,3-PDO production was not enhanced. quired for 1,3-PDO production. The decrease in 1,3-PDO formation in Mutation of dhaD and dhaK with coexpression of native dhaB123 and the lactate/2,3-BDO deficient strain was the result of pyruvate accu- dhaT resulted in abolished production of 2,3-BDO, lactate, and ethanol, mulation, which can interfere with glycerol dissimilation in 2,3-BDO but the 1,3-PDO titer and glycerol consumption were reduced relative deficient mutants (Lee et al., 2014). to the wild-type strain (Horng et al., 2010). In the absence of a The major side metabolites produced during glycerol fermentation functional glp regulon for respiratory dissimilation of glycerol, the in Klebsiella sp. are lactate, 2,3-BDO, ethanol, succinate, and acetate, oxidative branch is the major pathway for fermentative glycerol and inactivation of these pathways can potentially enhance 1,3-PDO metabolism in K. pneumoniae, such that inactivation of the dha production. Formation of acetate and 2,3-BDO during glycerol fer- regulon potentially hinders glycerol dissimilation and reduces NADH mentation for 1,3-PDO production results in a surplus of 2 mol and 0.5 levels. Activating the glp regulon in K. pneumoniae via overexpression of mol NADH, respectively, per mol glycerol consumed, while formation native glpK significantly altered cellular metabolism with acetoin of lactate and ethanol is redox balanced and formation of succinate production being favored over reductive glycerol metabolism (Wang generates a deficit of 1 mol NADH per mol glycerol consumed. While et al., 2017b), while reduced expression of glpK may mildly stimulate the formation of each metabolite, which is accompanied by ATP pro- glycerol dissimilation and improve 1,3-PDO production under suitable duction for cell growth and maintenance, can relieve pyruvate accu- aeration conditions. In parallel, expression of formate dehydrogenase mulation resulting from the compromised tricarboxylic acid (TCA) (FDH) and/or pyridine nucleotide transhydrogenase (UDH) can cycle in Klebsiella sp. (Cabelli, 1955), reduced carbon flux toward 1,3- potentially regenerate NADH to restore the redox balance during 1,3- PDO formation may occur. Inactivating the ethanol pathway in K.

545 A.W. Westbrook et al. Biotechnology Advances 37 (2019) 538–568

Table 2 Summary of literature for Klebsiella sp. strains engineered for value-added chemical production from glycerol.

Parent strain Product Genetic strategy Titer (g/L) Cultivation Reference mode

Kp ZG25 1,3-PDO Mutate budC to reduce 2,3-BDO formation 67 fedbatch (Guo et al., 2013) Kp TUAC01 1,3-PDO Express dhaT 20 batch (Hao et al., 2008b) Kp KG2 (lacks ldhA) 1,3-PDO Mutate poxB and pta-ackA to reduce acetate formation 77 fedbatch (Lin et al., 2016) Kp J2B ΔldhA 1,3-PDO Mutate budA, budB, budC, or all three to reduce 2,3-BDO formation 31 fedbatch (Kumar et al., 2016) Kp KG1 1,3-PDO 45 fedbatch (Zhao et al., 2009) Express dhaD and dhaT to elucidate their roles during microaerobic cultivation Kp AK (lacks oxidative 1,3-PDO 26 fedbatch (Luo et al., 2013) pathway) Express dhaB123, dhaT, or yqhD, or dhaT and yqhD, or dhaB123 and dhaT (along with gdrAB) Kp Cu 1,3-PDO -Mutate dhaD and dhaK while inserting the lacZ promoter (E. coli) 5 flask (Seo et al., 2009) upstream of dhaB123 to interrupt oxidative metabolism without disturbing the reductive branch -Mutate dhaR to interrupt oxidative metabolism -Coexpress dhaT and gdrAB Kp ΔldhA 1,3-PDO 68 fedbatch (Lee et al., 2014) -Mutate budB and pta to reduce 2,3-BDO and acetate formation, respectively -Coexpress pdc and aldB (Z. mobilis) to stimulate conversion of pyruvate to ethanol to restore metabolism in ΔbudB mutant Kp CICIM B0057 1,3-PDO Express yqhD (E. coli) and/or dhaT 18 batch (Zhuge et al., 2010) Ko M5aI 1,3-PDO Mutate ldhA to reduce lactate formation 63 fedbatch (Yang et al., 2007) Kp AK and Cu 1,3-PDO Express yqhD to increase NADH levels 8 flask (Seo et al., 2010) Kp WT 1,3-PDO -Mutate budC to reduce 2,3-BDO formation 72 fedbatch (Wu et al., 2013) -Chromosomally express fdh (P. pastoris) to regenerate NADH Kp KG2 and KG3 1,3-PDO 69 fedbatch (Zhu et al., 2015)

Express puuC via leakage from Ptac promoter to enhance 3-HP and NADH formation Kp YMU2 1,3-PDO Mutate aldA to reduce ethanol formation 71 fedbatch (Zhang et al., 2006a, 2006b) Kp WT 1,3-PDO -Mutate dhaD and dhaK to interrupt oxidative metabolism 8 flask (Horng et al., 2010) -Coexpress dhaT and dhaB123 Ko M5aI 1,3-PDO Mutate budA to reduce 2,3-BDO formation 27 fedbatch (Zhang et al., 2012) Kp HR526 1,3-PDO Mutate ldhA to reduce lactate formation 102 fedbatch (Xu et al., 2009) Kp Cu 1,3-PDO -Mutate budB and ldhA to reduce 2,3-BDO and lactate formation, 103 fedbatch (Oh et al., 2012b) respectively Kp KG1 1,3-PDO Coexpress dhaD and dhaT 17 flask (Zhao et al., 2009) Kp ACCC 10082 1,3-PDO Coexpress dhaD and dhaT 59 fedbatch (Chen et al., 2009) Kp 2-1 1,3-PDO -Mutate ldhA and aldH to reduce lactate and ethanol formation, 88 fedbatch (Chen et al., 2016) respectively Kp CICIM B0057 1,3-PDO 22 flask (Lu et al., 2016) Reduce 2,3-BDO formation by reducing expression of budA, budB, and budC via asRNA Kp CGMCC 1.6366 1,3-PDO 16 batch (Zhou et al., 2017a, -Mutate pflB or acoABCD to reduce acetate formation 2017b) Kp KG2 1,3-PDO -Mutate pck or ppc to reduce succinate formation 72 fedbatch (Zhang et al., 2017) Kp KCTC 2242 1,3-PDO -Introduce point mutations in DhaT via error prone PCR to increase 86 fedbatch (Wang et al., 2017c) activity -Mutate crr to relieve catabolite repression for glycerol/glucose co- fermentation to increase biomass and NADH levels -Overexpress glpF to enhance glycerol uptake -Coexpress fdh (P. pastoris), gdh (B. subtilis), and udh to regenerate NADH Kp ATCC 200721 1,3-PDO 49 fedbatch (Oh et al., 2018) ΔldhA -Mutate budA, budB, or budC to reduce 2,3-BDO formation Kp WT 1,3-PDO 78 fedbatch (Lu et al., 2018) -Mutate arcA to increase expression of enzymes in the TCA cycle -Mutate ldhA to reduce lactate formation -Mutate crr to relieve catabolite repression for glycerol/glucose co- fermentation to increase biomass and NADH levels Kp KCTC 2242 1,3-PDO -Mutate ldhA, pflB, and budA to reduce lactate, acetate, and 2,3-BDO 21 batch (Lee et al., 2018) ΔwabG formation, respectively -Mutate dhaD and glpK to prevent glycerol assimilation into biomass -Modify the 5’-UTR of mtlA to improve the efficiency of mannitol usage -Mutate dhaK123 and overexpress dhaK2 to enhance expression of dhaT Ko PDL-0(WT) 1,3-PDO/ -Mutate budA and budB, adhE, and ackA-pta to reduce 2,3-BDO, 70 / 100 (D- fedbatch (Xin et al., 2017) lactate ethanol, and acetate formation, respectively or L-lactate) -Replace ldhD with ldhL (L. casei) to facilitate L-lactate production Kp KCTC12133BP 1,3-PDO/2,3- 125 fedbatch (Park et al., 2017) BDO -Mutate ldhA and mdh to reduce lactate and succinate formation, respectively (continued on next page)

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Table 2 (continued)

Parent strain Product Genetic strategy Titer (g/L) Cultivation Reference mode

Kp KCTC 2242 ΔldhA ΔptsG 1,3-PDO/2,3- 79 / 32 fedbatch (Wang et al., 2017d) BDO -Construct EDP from Z. mobilis and coexpress udh to regenerate NADH -Coexpress dhaT Kp DSMZ 2026 3-HP/ 16 / 17 fedbatch (Ashok et al., 2011) 1,3-PDO Express puuC and mutate dhaT to balance 3-HP and 1,3-PDO production Kp WM3 1,3-PDO/ 24 / 49 fedbatch (Huang et al., 2012) 3-HP Express ALDHs from Zymomonas mobilis (adhB), Lactobacillus collinoides (aldH and pduQ), K. pneumoniae (aldA, aldB, puuC, ydcW, etuE, feaB, gabD and badH), or E. coli (aldA, aldH and ycdW) Kp J2B 1,3-PDO/ Express KGSADH (A. brasilense) 16 / 12 fedbatch (Kumar et al., 2012) 3-HP Kp J2B 1,3-PDO/ -Express KGSADH (A. brasilense) 23 / 23 fedbatch (Kumar et al., 2013) 3-HP -Mutate ldhA to reduce lactate formation Kp DSMZ 2026 1,3-PDO/ Express dhaS (B. subtilis) 18 / 27 fedbatch (Su et al., 2015) 3-HP Kp AK 1,3-PDO/ Express aldH, dhaT amd gdrAB in mutant defective in oxidative 23 / 7 fedbatch (Luo et al., 2011a, 3-HP glycerol metabolism 2011b) Kp J2B 1,3-PDO/ 21 / 43 fedbatch (Ko et al., 2017) 3-HP - Mutate ldhA, adhE, frdA, and pta-ackA to reduce lactate, ethanol, succinate, and acetate formation, respectively -Mutate glpK or dhaD to reduce acetate without disrupting pta-ackA to improve glycerol consumption -Overexpress dhaT, or dhaB123 and gdrAB Kp J2B 3-HP 16 fedbatch (Ko et al., 2012) -Mutate dhaT and yqhD to reduce 1,3-PDO formation -Express puuC, KGSADH (A. brasilense) or aldH (E. coli) Kp J2B ΔdhaT ΔyqhD 3-HP Mutate ahpF and adhE in a ΔdhaT ΔyqhD double mutant expressing 9 fedbatch (Ko et al., 2015) KGSADH (A. brasilense) to reduce 1,3-PDO formation Kp DSMZ 2026 3-HP 23 fedbatch (Ashok et al., 2013a) -Mutate glpK to force glycerol through the fermentative pathway in the presence of nitrate, -Mutate dhaT to reduce 1,3-PDO formation -Overexpress puuC Kp DSMZ 2026 3-HP 84 fedbatch (Li, Y. et al., 2016)

-Optimize puuC expression through promoter selection (Ptac) -Mutate ldh1 and ldh2, and pta to reduce lactate and acetate formation, respectively Kp DSMZ 2026 3-HP 28 fedbatch (Ashok et al., 2013b) -Coexpress dhaB123, gdrAB and puuC in a ΔdhaT ΔyqhD double mutant Ko M1 2,3-BDO 132 fedbatch (Cho et al., 2015) -Mutate pduC and ldhA to reduce 1,3-PDO and lactate formation, respectively Kp WT 1-butanol -Express ter (T. denticola), and bdhA and bdhB (C. acetobutylicum) 0.029 flask (Wang et al., 2014a, -Express kivd (L. lactis) to facilitate the 2-keto acid pathway 2014b, 2014c) -Reduce 1,3-PDO formation by reducing expression of dhaB123, dhaT, and/or gdrAB via asRNA -Reduce 2,3-BDO formation by reducing expression of budB and/or budC via asRNA Kp WT 1-butanol 0.1 flask (Wang et al., 2015a, -Express kivd (L. lactis), leuABCD, and adhe1 (C. acetobutylicum) 2015b) -Enhance NADH regeneration via expression of fdh (P. pastoris), gdh, or udh -Reduce 1,3-PDO formation by reducing expression of dhaB123, dhaT, and/or gdrAB via asRNA Kp 13 1-butanol 0.2 flask (Wang et al., 2017a) -Express kivd (L. lactis), leuABCD, and adhe1 (C. acetobutylicum) -Repress expression of metA, ilvI, ilvB, and alaA via CRISPRi to increase carbon flux toward 1-butanol production Kp ATCC 200721 ΔldhA 2-butanol 0.3 batch (Oh et al., 2014) -Express ilvIH, ilvD, and kivd and adhA (L. lactis) -Mutate budA to reduce 2,3-BDO formation GEM167 ethanol 31 fedbatch (Oh et al., 2012a) -Mutate ldhA to reduce lactate formation -Coexpress pdc and adhII (Z. mobilis) ATCC 25955 D-lactate 142 fedbatch (Feng et al., 2014) -Overexpress ldhA -Mutate dhaT and yqhD to reduce 1,3-PDO formation ATCC 25955 acetoin -Mutate budC, gldA and dhaD to prevent conversion of acetoin to 2,3- 32 fedbatch (Wang et al., 2017b) BDO (continued on next page)

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Table 2 (continued)

Parent strain Product Genetic strategy Titer (g/L) Cultivation Reference mode

-Express glpK to activate silent glp operon -Express nox (L. brevis) to increase NAD+/NADH ratio ATCC 25955 P(3HP) -Express dhaB123, gdrAB, and aldH (E. coli) to enhance 3-HP 0.2 flask (Feng et al., 2015) formation -Express prpE and phaC to convert 3-HP to P(3HP) -Mutate dhaT and yqhD to reduce 1,3-PDO formation ackA, acetate kinase; acoABCD, pyruvate dehydrogenase complex; adhII, aldehyde/alcohol dehydrogenase; adhA, alcohol dehydrogenase; aldB, aldehyde dehydrogenase; adhE, bifunctional acetaldehyde-CoA/alcohol dehydrogenase; alaA, alanine transaminase; ahpF, alkyl hydroperoxide oxidoreductase; aldH, aldehyde dehydrogenase; bdhA, butanol dehydrogenase A; bdhB, butanol dehydrogenase B; budA, α-acetolactate decarboxylase; budB, α-acetolactate synthase; budC, 2,3-BDO dehydrogenase/acetoin reductase; dhaB123, glycerol dehydratase; crr, glucose phosphotransferase protein; dhaD, glycerol dehydrogenase; dhaK, dihydroxyacetone kinase; dhaR, transcriptional activator of the dha regulon; dhaS, aldehyde dehydrogenase; dhaT, NADH-dependent 1,3-PDO oxidoreductase; fdh, formate dehydrogenase; frdA, succinate dehydrogenase; gdh, glucose dehydrogenase (Bacillus subtilis); gdrAB, glycerol dehydratase reactivation factor; gldA, glycerol dehydrogenase; glpF, glycerol transporter; glpK, glycerol kinase; ilvB, acetolactate synthase isozyme 1 large subunit; ilvD, dihydroxyacid dehydratase; ilvI, acetolactate synthase isozyme 3 large subunit; ilvIH, keto-acid reducto-isomerase; KGSADH, α-ketoglutaric semialdehyde dehydrogenase; kivD, α-ketoisovalerate decarboxylase; lacZ, β-galactosidase; ldh1, lactate dehydrogenase; ldh2, lactate dehydrogenase; ldhA, D-lactate dehydrogenase; ldhD, D-lactate dehydrogenase; ldhL, L-lactate dehydrogenase; leuA, 2-isopropylmalate synthase; leuB, 3-isopropylmalate dehydrogenase; leuCD, 3-isopropylmalate dehydratase subunits C and D; mdh, malate dehydrogenase; metA, pck, carboxykinase; nox, NADH oxidase; pdc, pyruvate decarboxylase; homoserine O-succinyltransferase; pduC, diol dehydratase large subunit; pflB, pyruvate formate lyase; phaC, polyhydroxyalkanoate synthase; poxB, pyruvate oxidase; ppc, phosphoenolpyruvate carboxylase; prpE, propionyl-CoA synthetase; pta, phosphotransacetylase; puuC, aldehyde dehydrogenase; ter, trans-2-enoyl-CoA reductase; udh, pyridine nucleotide transhydrogenase; yqhD, NADPH-dependent 1,3-PDO oxidoreductase; other terms: ALDH, aldehyde dehydrogenase; 1,3-PDO, 1,3-propanediol; 2,3-BDO, 2,3-butanediol; 3-HP, 3-hydroxypropionic acid; asRNA, antisense RNA; CRISPRi, Clustered Regularly Interspaced Palindromic Repeats interference; EDP, Entner-Doudoroff pathway; Ko, Klebsiella oxytoca; Kp, Klebsiella pneumoniae; NADH, nicotinamide adenine dinucleotide; P(3HP), poly(3-hydroxypropionate); PCR, polymerase chain reaction.

pneumoniae via mutation of aldA (encoding ALDH; AldA) drastically Moreover, mutation of budC modestly increased and decreased the 1,3- reduced ethanol formation, resulting in significant increases in the yield PDO and 2,3-BDO titers, although mutation of the entire bud operon and titer of 1,3-PDO in a fedbatch fermentation, despite decreased (i.e. budABC) resulted in the lowest 1,3-PDO titer. While the culture glycerol consumption and biomass accumulation. (Zhang et al., 2006a, performance of the ΔbudABC mutant was significantly improved, i.e. 2006b). On the other hand, mutation of aldH (encoding ALDH; AldH) production of the target metabolite (1,3-PDO) increased, by increasing and ldhA (encoding lactate dehydrogenase; LdhA) abolished ethanol the buffering capacity of the medium in a shake flask, the 1,3-PDO titer and lactate formation, respectively, although the 1,3-PDO titer was declined significantly due to excessive pyruvate accumulation during a marginally improved (Chen et al., 2016). Similarly, inactivation of ldhA fedbatch fermentation (Kumar et al., 2016), indicating inefficient op- significantly increased and decreased NADH and lactate levels, re- eration of the TCA cycle. Similarly, reduced expression of budA and spectively, leading to substantial 2,3-BDO and ethanol formation, and budB via antisense RNA (asRNA)-mediated transcriptional interference the 1,3-PDO titer increased slightly in a fedbatch fermentation (Xu significantly decreased 2,3-BDO production without substantially af- et al., 2009). In contrast to K. pneumoniae, mutation of ldhA in Klebsiella fecting the 1,3-PDO titer, and repression of budC expression did not oxytoca slightly enhanced glycerol dissimilation, abolished lactate appreciably affect culture performance (Lu et al., 2016). In contrast, production, and significantly increased the 1,3-PDO titer, however, 2,3- mutation of budA, budB, or budC in a ∆ldhA mutant reduced 2,3-BDO BDO formation markedly increased (Yang et al., 2007). and 1,3-PDO production, with the ΔldhA ΔbudA double mutant pro- Elevated 2,3-BDO levels can be particularly problematic since its ducing significantly less 1,3-PDO than all other strains (Oh et al., 2018). boiling point is similar to that of 1,3-PDO, making downstream pur- To reduce pyruvate accumulation through increased ethanol synthesis, ification more difficult (Xiu and Zeng, 2008), and considerable effort pdc and aldB, encoding pyruvate decarboxylase (PDC) and ALDH (AldB) has been made to alleviate 2,3-BDO production. Mutation of ldhA and of Zymomonas mobilis, respectively, were coexpressed in a ΔldhA ΔbudB budB, encoding α-acetolactate synthase (BudB), significantly decreased double mutant (Oh et al., 2012b), resulting in restored glycerol dis- lactate and 2,3-BDO formation, respectively, although the 1,3-PDO titer similation and 1,3-PDO production. These studies underscore the deli- was also reduced (Oh et al., 2012b). Inactivation of budC, encoding 2,3- cate balance between 1,3-PDO and 2,3-BDO production in Klebsiella sp., BDO dehydrogenase/acetoin reductase (BudC), moderately decreased and further suggest the unstable nature of the TCA cycle, which is a and increased 2,3-BDO and 1,3-PDO production, respectively (Guo critical issue to be addressed. et al., 2013). The moderate reduction in 2,3-BDO production in cultures Inactivating acetate pathways to enhance 1,3-PDO production has of the ΔbudC mutant is likely due to the promiscuity of DhaD, which can also been explored. Mutation of pta (encoding phosphotransacetylase; catalyze the interconversion of acetoin and 2,3-BDO in response to PTA) and ackA (encoding acetate kinase; AckA) restored cell growth, elevated intracellular NADH levels during glycerol catabolism (Wang reduced acetate production, and modestly increased the 1,3-PDO titer et al., 2014a, 2014b, 2014c), while an alternate/complementary ex- under the ΔldhA ΔpoxB background (Lin et al., 2016). Similarly, mu- planation is the presence of the 2,3-BDO replenishment cycle in K. tation of pflB, encoding PFL (PflB), decreased acetate production but pneumoniae (Syu, 2001). Mutation of budA, encoding α-acetolactate did not significantly improve the 1,3-PDO titer, while mutation of decarboxylase (BudA), in K. oxytoca abolished 2,3-BDO formation and acoABCD, encoding the PDH complex (AcoABCD), increased the pro- moderately increased the 1,3-PDO titer, although the acetate and lac- ductivity but decreased the yield of 1,3-PDO due to significantly in- tate titers increased substantially (Zhang et al., 2012). Similarly, mu- creased formate and ethanol formation (Zhou et al., 2017a, 2017b). tation of budA or budB in a K. pneumoniae ΔldhA mutant abolished 2,3- Finally, in an attempt to alleviate succinate formation, pck (encoding BDO formation, although the ΔldhA ΔbudA double mutant produced PEP carboxykinase; PCK) was inactivated, resulting in improved growth significantly less 1,3-PDO than the parent strain (Kumar et al., 2016). and reduced acetate and 2,3-BDO formation, albeit without significant

548 A.W. Westbrook et al. Biotechnology Advances 37 (2019) 538–568 changes in 1,3-PDO or succinate production, and mutation of ppc (en- encoding enzymes of the TCA cycle in response to decreasing levels of coding PEP carboxylase; PPC) did not affect culture performance reduced quinones (Bekker et al., 2010; Shalel-Levanon et al., 2005). (Zhang et al., 2017). A considerable body of literature suggests that Mutation of arcA in K. pneumoniae resulted in a ≥9-fold increase in the completely abolishing side metabolite formation is not feasible in transcription of gltA (encoding citrate synthase; GltA), icd (encoding Klebsiella sp., potentially due to their incomplete TCA cycle that can isocitrate dehydrogenase; ICD), sdhC, and sucC (encoding succinyl-CoA lead to accumulation of inhibitory levels of pyruvate (Kumar et al., synthetase β subunit; SucC), resulting in a significant decrease in 2016). Prior work indicates that the flavin adenine dinucleotide (FAD) acetate accumulation and the intracellular NAD+/NADH ratio, and an binding subunit of the succinate:quinone oxidoreductase complex, en- increase in the specific 1,3-PDO titer (Lu et al., 2018). However, lactate coded by sdhA (SdhA), and fumarase A, encoded by fumA (FumA), production increased substantially, which was resolved by mutating which successively convert succinate to fumarate and fumarate to ldhA in the ΔarcA mutant, resulting in complete cessation of lactate malate in the TCA cycle, are weakly expressed in K. pneumoniae accumulation and a slight increase in the 1,3-PDO titer. In the presence (Cabelli, 1955; Kumar et al., 2016). Fumarase A and fumarase B (en- of glucose or other preferred carbon sources, EIIAGlc is in the depho- coded by fumB; FumB) contain [4Fe-4S] clusters that are sensitive to sphorylated state and will bind to GlpK to inhibit glycerol uptake oxidative damage, while fumarase C (encoded by fumC; FumC) is in- (Deutscher et al., 2006). Accordingly, mutation of crr in the ΔarcA sensitive to oxygen (Liochev and Fridovich, 1993). Accordingly, ex- ΔldhA double mutant enabled co-fermentation of glucose and glycerol, pression of the appropriate fumarase isozyme from E. coli, selected resulting in a moderate increase in the 1,3-PDO titer, albeit with re- based on the aeration conditions employed for glycerol fermentation, duced rates of cell growth and glycerol consumption, and increased may activate the TCA cycle in Klebsiella sp., in turn, reducing side acetate and lactate production (Lu et al., 2018). Similarly, mutation of metabolite formation and promoting glycerol dissimilation. Similarly, crr in K. pneumoniae moderately improved 1,3-PDO production, al- expression of the succinate:quinone oxidoreductase complex, encoded though, in this case, side metabolite formation was reduced (Wang by sdhABCDE (SdhABCDE), may also improve culture performance. It et al., 2017c). Also, co-fermentation of glycerol and untreated molasses may be necessary to perform more extensive transcriptional analysis of significantly improved the 1,3-PDO titer inaΔldhA Δcrr double mutant genes encoding enzymes comprising the TCA cycle during glycerol (Oh et al., 2013a). Moreover, overexpression of native glpF significantly fermentation to identify other gene targets in Klebsiella sp. for metabolic enhanced the glycerol consumption rate and modestly increased the manipulation. 1,3-PDO titer in a fedbatch fermentation (Wang et al., 2017c). Finally, Coproduction of 1,3-PDO with a second major metabolite is an al- expression of fdh, encoding FDH from Candida boidinii, in K. oxytoca, ternate approach to completely eliminating side metabolite production. slightly increased the 1,3-PDO titer while ethanol production increased 1,3-PDO/lactate co-production is a redox balanced process that can substantially (Zhang et al., 2009). However, the intracellular NAD+/ result in high yields of both metabolites in K. oxytoca (Xin et al., 2017). NADH ratio was not affected by the activity of the NADH regeneration 2,3-BDO, ethanol, and acetate formation were either abolished or sig- system, as was the case in E. coli (Berrı,́ S.J., San, K.-Y., Bennett, G., nificantly reduced via mutation of budA and budB, adhE (encoding al- 2002), suggesting that either K. oxytoca can efficiently recycle NADH dehyde/alcohol dehydrogenase; AdhE), and ackA-pta, respectively, through native pathways, expression levels of fdh were not sufficient to leading to a high 1,3-PDO/D-lactate yield of 0.95 mol/molglycerol. affect NADH regeneration, or substantial conversion of formate2 toH Moreover, optically pure L-lactate was co-produced with 1,3-PDO at a and CO2 by the formate hydrogen lyase (FHL) complex occurred. Si- similar yield by replacing the native D-LDH (encoded by ldhD) with L- milarly, chromosomal insertion of fdh from Pichia pastoris in the budC LDH from Lactobacillus casei (encoded by ldhL)(Xin et al., 2017). High- locus of K. pneumoniae significantly increased biomass accumulation level 1,3-PDO/2,3-BDO co-production from glycerol was recently de- and decreased 2,3-BDO production, although the 1,3-PDO titer was monstrated in K. pneumoniae. 1,3-PDO/2,3-BDO co-production was modestly improved (Wu et al., 2013), while coexpression of fdh from P. significantly improved in K. pneumoniae via mutation of ldhA, while pastoris, gdh (encoding glucose dehydrogenase; GluDH) from Bacillus mutation of mdh, encoding malate dehydrogenase (MDH), significantly subtilis, and native udh increased both 1,3-PDO and lactate production reduced succinate formation in the ΔldhA mutant with only a modest (Wang et al., 2017c). improvement in the 1,3-PDO/2,3-BDO yield (Park et al., 2017). Con- Recently, multiple parallel strain engineering strategies led to a very struction of the Entner-Doudoroff pathway (EDP) from Z. mobilis in K. high 1,3-PDO yield of 0.76 mol/molglycerol in a K. pneumoniae strain in pneumoniae with coexpression of native dhaT and udh (encoding UDH) which wabG, encoding a protein involved in the transfer of galacturonic significantly improved 1,3-PDO/2,3-BDO co-production during gly- acid (WabG), was mutated to prevent lipopolysaccharide synthesis (Lee cerol/glucose co-fermentation (Wang et al., 2017d). This strategy et al., 2018). Mutation of ldhA and pflB in the ∆wabG mutant abolished aimed to increase NADPH levels via glucose metabolism through the lactate and acetate production, respectively, with modest increases in EDP from Z. mobilis with subsequent regeneration of NADH from the 1,3-PDO titer, while mutation of budA nearly eliminated 2,3-BDO NADPH via native UDH (Wang et al., 2017d). However, 1,3-PDO/2,3- production at the expense of increased acetate production and severely BDO co-production may be practically infeasible due to the difficulty reduced glycerol consumption and 1,3-PDO production. To prevent associated with separating these compounds, and carbon loss is sig- glycerol assimilation into biomass, dhaD and glpK were mutated in the nificant during 2,3-BDO production due to the successive decarbox- ∆wabG ∆ldhA ∆pflB ∆budA quadruple mutant, and the resulting strain ylations from pyruvate to acetoin (Fig. 1). KMK-23 was cultured with glycerol and glucose as co-substrates to Other strain engineering strategies to improve 1,3-PDO production enable growth on glucose with glycerol being converted to 1,3-PDO at a from glycerol include protein engineering of key enzymes, globally significantly higher yield than the parent strain (Lee et al., 2018). upregulating the expression of enzymes in the TCA cycle, relieving Subsequently, mannitol was selected as the most suitable co-substrate catabolite repression to enable co-fermentation of glycerol and glucose, with glycerol, and the 5’- untranslated region (UTR) of mtlA, encoding the expression of glycerol transporters to enhance glycerol uptake, and the mannitol-specific transporter (MtlA), was modified in the genome of the expression of enzymes that can regenerate NADH. Transformation KMK-23 to improve the efficiency of mannitol utilization, which, in of integration cassettes generated with error prone PCR resulted in turn, improved cell growth and 1,3-PDO production in the resulting point mutations to DhaT (D41G and H57Y) that moderately increased strain KMK-23M. Finally, based on the observation that DhaL, i.e. a its activity and improved 1,3-PDO production (Wang et al., 2017c). homologue of the subunit of DhaKII encoded by dhaK2, positively Creating large mutant libraries via error prone PCR, or rational protein regulates DhaR, in turn, increasing the expression of DhaT in E. coli engineering may be effective strategies to further enhance the activity (Bächler et al., 2005), dhaK123 was mutated in KMK-23M, resulting in of DhaT, or to relieve the inhibition of DhaT by 3-HPA. The ArcA-ArcB strain KMK-46, followed by overexpression of dhaK2 in KMK-46, re- two component system negatively regulates the expression of genes sulting in strain KMY, which produced significantly more 1,3-PDO than

549 A.W. Westbrook et al. Biotechnology Advances 37 (2019) 538–568

KMK-23M. Interestingly, 1,3-PDO production was abolished in KMK-46, increased the 3-HP titer significantly in the wild-type strain and could not be rescued by simply overexpressing dhaR. Moreover, the overexpressing puuC, while 3-HP production was highest in cultures overexpression of dhaT in KMK-23M or KMK-46 resulted in significantly of a ΔglpK ΔdhaT double mutant supplemented with nitrate as an lower 1,3-PDO production, compared to KMY (Lee et al., 2018), sug- electron acceptor (Ashok et al., 2013a). Subsequently, inactivation of gesting the complex and poorly understood regulation of glycerol me- dhaT and yqhD neither eliminated 1,3-PDO production nor substantially tabolism in K. pneumoniae. increased 3-HP formation in the wild-type strain, while coexpression of native puuC, dhaB123, and gdrAB significantly increased 3-HP 3.1.2.2. 3-HP. Due to its use in the synthesis of many industrially production in the ΔdhaT ΔyqhD double mutant, relative to puuC important chemicals, 3-HP has received increasing attention as a target overexpression alone, during fedbatch operation (Ashok et al., metabolite in engineered Klebsiella sp. 3-HP production from glycerol 2013b). Further efforts to reduce 1,3-PDO formation for enhanced 3- generates 1 mol NADH per mol glycerol consumed, such that co- HP production in a ΔdhaT ΔyqhD double mutant expressing KGSADH production with 1,3-PDO is favorable due to redox balance, or an from A. brasilense via mutation of adhE and ahpF, encoding alkyl exogenous electron acceptor can improve the 3-HP/1,3-PDO selectivity hydroperoxide oxidoreductase (AhpF), were unsuccessful (Ko et al., (Ashok et al., 2013a). Strain engineering strategies employed to 2015), suggesting that additional 1,3-PDOORs have yet to be identified. enhance the marginal levels of naturally produced 3-HP primarily Finally, the 3-HP titer increased by 3.9-fold and the 1,3-PDO titer focused on enhancing 3-HPA conversion to 3-HP via expression of decreased substantially upon expression of dhaS, encoding ALDH from ALDHs, and inactivating 1,3-PDOORs to increase available 3-HPA. B. subtilis (DhaS), but with significantly reduced glycerol consumption Balanced 3-HP/1,3-PDO co-production was achieved via and biomass accumulation (Su et al., 2015). overexpression of native puuC and mutation of dhaT and, relative to For more effective 3-HP production, the strain engineering strate- the parent strain, the 3-HP titer was improved by more than 10-fold gies described above can be integrated with bioprocessing strategies. with a similar 1,3-PDO titer (Ashok et al., 2011). The existence of For example, while overexpressing native puuC from the strong tac putative oxidoreductases which convert 3-HPA to 1,3-PDO is well promoter, the 3-HP titer increased by 6-fold relative to that obtained documented in Klebsiella sp., making elimination of 1,3-PDO under the lac promoter, and an extremely high 3-HP titer of 84 g/L was formation difficult (Ko et al., 2015; Ko et al., 2012). Similarly, obtained with a high 3-HP/1,3-PDO selectivity by manipulating aera- overexpression of native puuC increased the 3-HP titer by 5.5-fold in tion conditions (Li et al., 2016a, 2016b). It has been demonstrated that a strain with an inactivated oxidative branch of fermentative glycerol the selectivity of 3-HP to 1,3-PDO is highly dependent on aeration metabolism, however, no effect was observed in the wild-type strain, conditions, with fully aerobic operation abolishing both metabolites suggesting that puuC overexpression was merely generating due to insufficient B12 synthesis, fully anaerobic conditions favoring compensatory NADH in light of disruption of oxidative glycerol 1,3-PDO formation, and microaerobic conditions being conducive to co- metabolism (Luo et al., 2011a, 2011b). The ydcW and aldH genes production (Ashok et al., 2011; Ashok et al., 2013b; Li et al., 2016a, from E. coli and native ydcW were selected out of 14 ALDHs evaluated 2016b; Su et al., 2015). While an improved 3-HP/1,3-PDO selectivity is to enhance 3-HP production (Huang et al., 2012). The 3-HP titer was achievable without disrupting 1,3-PDOOR expression, 1,3-PDO pro- increased by 10-fold in a fedbatch fermentation of the aldH-expressing duction remains significant such that identification and mutation of strain, while the 1,3-PDO titer declined somewhat (Huang et al., 2012). additional 1,3-PDOORs may improve culture performance for 3-HP Similarly, expression of heterologous α-ketoglutaric semialdehyde production. Advanced genome engineering technologies such as the dehydrogenase from Azospirillum brasilense, encoded by KGSADH, CRISPR-Cas9 (Clustered Regularly Interspaced Palindromic Repeats- significantly increased both the 3-HP and 1,3-PDO titers, relative to CRISPR-associated [protein] 9) system facilitate multiplexed gene mu- the overexpression of native puuC, in a fedbatch fermentation of the tations in various microbial hosts (Jiang et al., 2015; Westbrook et al., ΔdhaT mutant (Ko et al., 2012). The dhaT mutation respectively 2016), and CRISPR interference (CRISPRi) has been applied to meta- increased and decreased 3-HP and 1,3-PDO production in strains bolic engineering in K. pneumoniae (Wang et al., 2017a). Accordingly, expressing puuC or KGSADH, while mutation of yqhD had no multiplexed mutation of genes encoding putative 1,3-PDOORs via the significant effect on culture performance. 3-HP/1,3-PDO co- CRISPR-Cas9 system may be a convenient and effective approach to production was subsequently investigated in a wild-type strain abolish 1,3-PDO production. Moreover, expression of NADH oxidase expressing KGSADH (Ko et al., 2017). Mutation of ldhA, adhE, and (NOX) can regenerate NAD+ to maintain the redox balance in a 1,3- frdA (encoding succinate dehydrogenase; FrdA) significantly reduced PDO-deficient mutant without sacrificing carbon flux to side metabolite lactate and ethanol formation, while succinate and 3-HP/1,3-PDO co- formation. production were only slightly affected. Due to a marked increase in acetate formation observed in the ΔldhA ΔadhE ΔfrdA triple mutant, pta- 3.1.2.3. Other products. High level 2,3-BDO production from glycerol ackA were mutated resulting in significantly decreased acetate was achieved in K. oxytoca through inactivation of pduC, encoding the formation, albeit at the expense of glycerol consumption and 3-HP DODHt large subunit, and ldhA (Cho et al., 2015). The 2,3-BDO titer production (Ko et al., 2017). Accordingly, acetate formation was reached 132 g/L in the ΔpduC ΔldhA double mutant, owing to reduced without disrupting pta-ackA by mutating glpK in the ΔldhA significantly increased glycerol dissimilation and decreased side ΔadhE ΔfrdA triple mutant, resulting in significantly increased glycerol metabolite formation, relative to the parent strain (Cho et al., 2015). consumption and 3-HP/1,3-PDO co-production, while mutation of dhaD Interestingly, the ratio of (2S,3S)-2,3-BDO to meso-2,3-BDO was higher impeded culture performance. Moreover, overexpression of dhaT, or for the ΔpduC ΔldhA double mutant, suggesting that it consumed more dhaB123 and gdrAB, increased and decreased 3-HP/1,3-PDO co- NADH per mole of 2,3-BDO produced than the parent strain. During production in the ΔldhA ΔadhE ΔfrdA triple mutant, and all genetic aerobic growth, a significantly lower intracellular+ NAD /NADH ratio strategies employed in this study either reduced or did not affect 3-HP was observed in a K. pneumoniae strain in which the NADH:quinone production (Ko et al., 2017). High-level 3-HP/1,3-PDO co-production oxidoreductase I complex (NDH-1) was inactivated, resulting in was also achieved via expression of KGSADH from A. brasilense in a markedly improved 2,3-BDO production without any significant resting cell system (Kumar et al., 2012), and mutation of ldhA could change in 1,3-PDO levels (Zhang et al., 2018). Strain engineering for significantly improve both 3-HP and 1,3-PDO titers (Ashok et al., 2,3-BDO production has recently been reviewed (Yang et al., 2017; 2013b). In contrast to the aforementioned ΔldhA ΔadhE ΔfrdA ΔglpK Yang and Zhang, 2018). quadruple mutant expressing KGSADH, a ΔglpK single mutant Production of 1-butanol from glycerol was achieved in K. pneumo- overexpressing native puuC produced less 3-HP relative to the wild- niae although titers were in the mg/L range (Wang et al., 2014a, 2014b, type strain expressing puuC. On the other hand, mutation of dhaT 2014c). The ter and bdhAB genes, encoding trans-2-enoyl-CoA reductase

550 A.W. Westbrook et al. Biotechnology Advances 37 (2019) 538–568 from Treponema denticola (Ter) and butanol dehydrogenases A (BdhA) production. On the other hand, activation of the silent glp regulon via and B (BdhB) from Clostridium acetobutylicum, respectively, were coexpression of native glpK further hindered glycerol dissimilation, but expressed to construct a modified CoA-dependent 1-butanol pathway, enabled acetoin production. Moreover, expression of nox, encoding while coexpression of kivD, encoding α-ketoisovalerate decarboxylase NOX from Lactobacillus brevis, significantly improved the acetoin titer, from Lactococcus lactis (KivD), to facilitate the 2-keto acid pathway which reached 32 g/L in a fedbatch fermentation (Wang et al., 2017b). significantly increased 1-butanol production. In addition, asRNAs were Finally, biopolymer production from glycerol was achieved in en- designed to target dhaB123, dhaT, gdrAB, budB, and budC to reduce the gineered K. pneumoniae (Feng et al., 2015). Poly(3-hydroxypropionate) formation of 1,3-PDO and 2,3-BDO, although silencing gdrB expression [P(3HP)], a polyhydroxyalkanoate (PHA) possessing high rigidity, alone was most effective in enhancing 1-butanol production (Wang ductility, and tensile strength in drawn films, is considered an attractive et al., 2014a, 2014b, 2014c). 1-Butanol production from glycerol in K. alternative to petroleum-based plastics (Andreeßen et al., 2010). P pneumoniae via the 2-keto acid pathway was significantly enhanced (3HP) production was achieved by first enhancing 3-HP biosynthesis through overexpression of native leuABCD (encoding 2-isopropylmalate through coexpression of native dhaB123 and gdrAB, and aldH from E. synthase, 3-isopropylmalate dehydrogenase, and 3-isopropylmalate coli. 3-HP was converted to 3-hydroxypropionyl-CoA (3-HP-CoA) which dehydratase subunits C and D), kivD from L. lactis, and adhE1 (encoding was subsequently polymerized into P(3HP) upon expression of prpE aldehyde/alcohol dehydrogenase; AdhE1) from C. acetobutylicum (encoding propionyl-CoA synthetase; PrpE) from E. coli, and phaC (Wang et al., 2015a, 2015b). Moreover, coexpression of fdh from P. (encoding PHA synthase; PhaC) from Ralstonia eutropha. The 3-HP titer Pastoris, and native udh and gdh to regenerate NADH, or simultaneous increased by 6.4-fold in a ΔdhaT ΔyqhD double mutant, though the transcription of asRNAs targeting the 1,3-PDO pathway markedly in- specific yield of P(3HP) was low, suggesting that 3-HP is not anideal creased the 1-butanol titer in K. pneumoniae, and side metabolite for- substrate for PrpE, which natively produces propionyl-CoA from pro- mation was greatly reduced (Wang et al., 2015a, 2015b). In addition, pionate (Feng et al., 2015). repression of expression of metA (encoding homoserine O-succinyltransferase; MetA), ilvI (encoding acetolactate synthase isozyme 3 large 3.2. Citrobacter sp. subunit; IlvI), ilvB (encoding acetolactate synthase isozyme 1 large subunit; IlvB), and alaA (encoding alanine transaminase; AlaA) via 3.2.1. Natural metabolism CRISPRi significantly increased 1-butanol production in K. pneumoniae, Citrobacter sp. are capable of fermentative glycerol dissimilation with repression of ilvB expression alone increasing the titer by 2.5-fold through coupled oxidative and reductive pathways (Fig. 1). Glycerol is (Wang et al., 2017a). A number of strain engineering strategies that oxidized to DHA by a soluble NAD+-dependent GlyDH (i.e. DhaD en- have been applied to enhance 1-butanol production in E. coli, for ex- coded by dhaD) and is subsequently phosphorylated into DHAP by ample (Atsumi et al., 2008; Dellomonaco et al., 2011; Dong et al., 2017; DhaK (encoded by dhaK)(Daniel and Gottschalk, 1992; Daniel et al., Ohtake et al., 2017), may also prove successful in Klebsiella sp. Mutation 1995). While ATP-dependent DhaK of Citrobacter freundii shows of pta can increase acetyl-CoA availability to drive acetoacetyl-CoA minimal homology to enzymes in other microorganisms (Daniel et al., production and simultaneously reduce acetate accumulation (Ohtake 1995), it contains K- and L-domains displaying similar functions to et al., 2017), and optimization of the ribosome binding site (RBS) DhaK and DhaL of E. coli, respectively (Siebold et al., 2003). Note that translation initiation rate of AdhE2 (Dong et al., 2017; Ohtake et al., enzymes encoded by the dha regulon (i.e. DhaB/DhaBCE, DhaD, DhaK, 2017), which is the most common bifunctional aldehyde/alcohol de- and DhaT) from Klebsiella sp. and Citrobacter sp. share 80-90% sequence hydrogenase employed for 1-butanol production, may enhance 1-bu- similarity, compared to 30-80% between Citrobacter sp. and Clostridium tanol production in Klebsiella sp. Moreover, disruption of the FHL sp. (Sun et al., 2003). In parallel to fermentative glycerol oxidation, complex, in combination with overexpression of fdh, can mitigate the glycerol is converted to 3-HPA by GDHt (i.e. DhaBCE encoded by redox imbalance imposed by 1-butanol production in Klebsiella sp. dhaBCE)(Seifert et al., 2001) and further reduced to 1,3-PDO by DhaT, (Dong et al., 2017). encoded by dhaT (Ainala et al., 2013). Analogous to DhaB in Klebsiella

Metabolic engineering of a ΔldhA K. pneumoniae mutant for 2-bu- sp., the activity of DhaBCE is dependent on B12, which is naturally tanol production from glycerol was explored through overexpression of synthesized in Citrobacter sp. (Keuth and Bisping, 1994). Upon the native ilvIH (encoding keto-acid reducto-isomerase; IlvIH) and ilvD conversion to 3-HPA, glycerol inactivates DhaBCE by disrupting the

(encoding dihydroxyacid dehydratase; IlvD), and kivD and adhA (en- cobalt-carbon (Co-C) σ-bond of the attached B12 (Seifert et al., 2001), coding alcohol dehydrogenase; AdhA) from L. lactis,(Oh et al., 2014). resulting in a closer binding of the modified coenzyme to the active site The 2-butanol titer increased significantly upon mutation of budA, and (Toraya, 2000). This altered dehydratase complex is reactivated by the ldhA mutation was essential for 2-butanol production which began DhaF and DhaG, encoded by dhaF and dhaG, respectively (Seifert et al., upon cessation of cell growth (Oh et al., 2014). 2001). In C. freundii, dhaF is located downstream of dhaBCE, whereas Enhanced ethanol production from glycerol was investigated in a K. dhaG is located downstream of dhaT with an opposite transcriptional pneumoniae mutant generated by γ-irradiation (Oh et al., 2011; Oh direction to dhaBCE (Seifert et al., 2001). These findings indicate that et al., 2012a). Coexpression of pdc and adhII (encoding aldehyde/al- dhaF is co-transcribed with dhaBCE, while dhaG is transcribed sepa- cohol dehydrogenase; AdhII) from Z. mobilis moderately increased the rately with dhaT to prevent accumulation of 3-HPA (Jiang et al., 2016; ethanol titer during fedbatch fermentation of crude glycerol (Oh et al., Seifert et al., 2001). 2011), while mutation of ldhA significantly improved ethanol production in the strain coexpressing pdc and adhII (Oh et al., 2012a). 3.2.2. Strain engineering High-level production of optically pure D-lactate from glycerol was Due to poor genetic tractability, Citrobacter sp. has been unpopular achieved by expressing native ldhA in a ΔdhaT ΔyqhD double mutant of as a host for bio-based production. However, some progress has been K. pneumoniae (Feng et al., 2014). Overexpression of ldhA with and made toward strain engineering of Citrobacter sp. for 1,3-PDO produc- without mutation of dhaT and yqhD increased the lactate titer by 11.5- tion. Preliminary attempts to produce 1,3-PDO in C. freundii focused on fold and 5.3-fold, respectively, and the lactate titer reached 142 g/L in a fermentation strategies using purified (Boenigk et al., 1993) or crude fedbatch fermentation under suitable aeration conditions (Feng et al., (Metsoviti et al., 2013) glycerol. As the first approach for strain en- 2014). gineering, expression of dhaT from Shimwellia blattae in C. freundii Acetoin production from glycerol has also been explored in en- significantly increased 1,3-PDO production compared to the wild-type gineered K. pneumoniae (Wang et al., 2017b). Mutation of budC, gldA strain during a fedbatch fermentation, with significant lactate forma- (encoding GlyDH; GldA), and dhaD prevented conversion of acetoin to tion observed as the cells entered the stationary phase (Celińska et al., 2,3-BDO, albeit with reduced glycerol consumption and no acetoin 2015). Accordingly, focus should be placed on minimizing lactate

551 A.W. Westbrook et al. Biotechnology Advances 37 (2019) 538–568 synthesis in C. freundii to further enhance 1,3-PDO production, as was glycerol under anaerobic conditions via coupled oxidative and re- successfully achieved in Citrobacter werkmanii (Maervoet et al., 2016). ductive branches of fermentative glycerol dissimilation, channeling C. werkmanii is also a native host for high-level 1,3-PDO production glycerol into glycolysis to generate energy and reducing equivalents (Maervoet et al., 2012a, 2012b) and preferentially utilizes glycerol for which are consumed through the production of reduced metabolites growth, even in the presence of multiple carbon sources (Maervoet such as 1,3-PDO (Tracy et al., 2012; Wang et al., 2015a, 2015b), with et al., 2014). Accordingly, dhaD was mutated to force dissimilated other acids and alcohols, including acetate, propionate, butyrate, lac- glycerol through the reductive pathway during co-fermentation with a tate, ethanol, and butanol being co-produced (Oh et al., 2014; Tracy second carbon source that could also support cell growth. Of several co- et al., 2012). Similar to glycerol dissimilation in other anaerobic mi- substrates tested, glycerol/glucose co-fermentation was considered fa- croorganisms, certain Clostridium sp. predominantly channel glycerol to vorable due to a moderate increase in the 1,3-PDO yield and the rela- glycolysis through the intermediates DHA and DHAP. As such, Clos- tively low cost of glucose, although insufficient NADH levels in the tridium sp. encode GlyDH (i.e. GldA or DhaD) and DHAK (i.e. DhaK). absence of glycerol oxidation resulted in 3-HPA accumulation Concurrently, glycerol is converted to 3-HPA and 1,3-PDO by GDHt (Maervoet et al., 2014). Moreover, ethanol, lactate, and acetate for- (encoded by dhaB, pduD, or pduE) and 1,3-PDOOR (encoded by dhaT), mation was significant during glycerol/glucose co-fermentation respectively, to maintain redox poise (Kubiak et al., 2012; Sandoval

(Maervoet et al., 2014), leading to subsequent inactivation of ldhA and et al., 2015; Sun et al., 2003). While GDHt is typically B12-dependent adhE to relieve the formation of these side metabolites (Maervoet et al., (Pyne et al., 2016a, 2016b; Saint-Amans et al., 2001; Siebold et al.,

2016). During glycerol/glucose co-fermentation, the ∆dhaD ∆adhE 2003), GDHt from C. butyricum (i.e. DhaB1 encoded by dhaB1) is B12- double mutant and ∆dhaD ∆ldhA ∆adhE triple mutant produced 2.2-fold independent, a phenomenon that appears to be uncommon in nature (Li and 2.7-fold more 1,3-PDO, respectively, compared to the ∆dhaD single et al., 2016a, 2016b). The completed genome sequence of C. diolis DSM mutant. In addition, 3-HPA accumulation was significantly reduced in 15410 also identified a putatively12 B -independent GDHt (Wang et al., the ∆dhaD ∆adhE double mutant, compared to the ∆dhaD single mutant, 2013a, 2013b). Interestingly, putative genes encoding enzymes in- and was undetectable in the ∆dhaD ∆ldhA ∆adhE triple mutant volved in glycerol dissimilation, i.e. glpK and glpD, have been identified (Maervoet et al., 2016). Lastly, mutation of arcA resulted in a sig- in several members of Clostridium, including Clostridium kluyveri, C. nificant increase in acetate, lactate, and succinate formation with 1,3- propionicum, and C. acetobutylicum (Nölling et al., 2001; Poehlein et al., PDO production being only slightly improved, whereas the ∆dhaD 2016; Seedorf et al., 2008). This is notable as these glycerol dissimila- ∆ldhA ∆arcA triple mutant did not produce any 1,3-PDO (Maervoet tion pathways are predominantly found in aerobic microorganisms. et al., 2016). As previously noted for Klebsiella sp., another potential Moreover, while various enzymes involved in glycerol dissimilation strategy to increase NADH levels for enhanced 1,3-PDO production in have been annotated in other Clostridium sp., these pathways appear to Citrobacter sp. is to simultaneously inactivate the FHL complex and be incomplete or dormant (Biebl, 2001; Sun et al., 2003; Vasconcelos overexpress FDH. In addition, 2,3-BDO formation has not been reported et al., 1994). Despite having genes associated with glycerol dissimila- in Citrobacter sp. engineered for enhanced 1,3-PDO production and this tion, most Clostridium sp. are unable to metabolize glycerol as the sole represents a major advantage over other natural 1,3-PDO producers carbon source, ostensibly due to the absence or deficiency of a 1,3-PDO such as Klebsiella sp. due to potentially reduced purification costs for pathway (González-Pajuelo et al., 2005; Seedorf et al., 2008). C. acet- separating 1,3-PDO and 2,3-BDO. obutylicum will utilize glycerol in the presence of a more oxidized Violacein, a blue-purple pigment possessing anti-bacterial, anti- carbon source such as glucose or pyruvate, or an exogenous electron viral, and anti-tumoral properties, is produced by diverse genera of acceptor such as methyl viologen or neutral red, suggesting that its bacteria, including Chromobacterium, Collimonas, Duganella, atypical glycerol dissimilation pathway is still active (Girbal et al., Janthinobacterium, Microbulbifer, and Pseudomonas (Choi et al., 2015; 1995; Girbal and Soucaille, 1994; Peguin et al., 1994; Vasconcelos Durán and Menck, 2001; Rettori and Durán, 1998). Biological pro- et al., 1994). duction of violacein has been relatively ineffective, with the most studied strain Chromobacterium violaceum generating small amounts of 3.3.2. Strain engineering crude violacein (Mendes et al., 2001; Sánchez et al., 2006). Expression Due to limited genetic tools for Clostridium sp. and the fact that of the violacein biosynthesis operon, i.e. vioABCDE, encoding L-tryp- glycerol is a recalcitrant carbon source under anaerobic conditions tophan oxidase (VioA), 2-imino-3-(indol-3-yl) propanoate dimerase (Jang et al., 2012; Pyne et al., 2014), strategies directed toward im- (VioB), deoxyviolaceinate synthase (VioC), protodeoxyviolaceinate proving glycerol utilization for the production of value-added meta- monooxygenase (VioD), and a protein of unknown function (VioE), bolites in Clostridium sp. remain uncommon. Random mutagenesis has from C. violaceum, resulted in negligible violacein production in en- been widely applied to improve glycerol dissimilation and, as such, gineered E. coli and Streptomyces albus (Pemberton et al., 1991; Sánchez mutants with improved phenotypes remain uncharacterized geneti- et al., 2006). On the other hand, expression of the violacein biosynth- cally. A C. pasteurianum mutant was capable of consuming glycerol at a esis operon from Duganella sp. in C. freundii resulted in a crude violacein rate almost double that of the parent strain, resulting in enhanced titer of 1.7 g/L from glycerol in shake flask cultures supplemented with production of 1,3-PDO and 1-butanol (Jensen et al., 2012). Similarly, a the precursor L-tryptophan (Jiang, P.-x., Wang, H.-s., Zhang, C., Lou, K., C. pasteurianum mutant with improved glycerol tolerance and growth Xing, X.-H., 2010), and the titer reached 4.1 g/L in a fedbatch culti- rate produced significantly more 1-butanol than the parent strain vation (Yang et al., 2011). (Malaviya et al., 2012), while 1-butanol tolerance and production increased in another mutant, even though glycerol consumption was not 3.3. Clostridium sp. affected (Gallardo et al., 2017). Genome shuffling was applied to C. diolis to improve 1,3-PDO tolerance, resulting in a marked increase in 3.3.1. Natural metabolism the 1,3-PDO yield with enhanced glycerol utilization compared to the Despite the ability to utilize a wide variety of carbon sources for parent strain (Otte et al., 2009). A C. pasteurianum mutant with im- Clostridium sp. (Tracy et al., 2012), natural catabolism of glycerol as the proved tolerance to crude glycerol was derived through random mu- sole carbon source appears to be confined to a small subset, of which C. tagenesis followed by directed evolution under increasing concentra- butyricum and C. pasteurianum are biotechnologically important. Ad- tions of crude glycerol, resulting in substantially increased 1-butanol ditionally, strains of Clostridium beijerinckii, Clostridium diolis, Clos- production and reduced 1,3-PDO formation, and the improved culture tridium saccharobutylicum, and Clostridium propionicum were also de- performance was found to be associated with a large deletion in spo0A, rived for glycerol dissimilation (Dobson et al., 2012; Tracy et al., 2012; encoding a transcriptional regulator for sporulation (SpoOA) (Sandoval Wang et al., 2014a, 2014b, 2014c). Clostridium sp. normally consume et al., 2015). In agreement with this work, mutation of spoOA

552 A.W. Westbrook et al. Biotechnology Advances 37 (2019) 538–568 significantly improved glycerol dissimilation with a concomitant in- substrates (Schütz and Radler, 1984; Talarico et al., 1990). Glycerol is crease in 1-butanol production (Schwarz et al., 2017). Mutation of dhaT converted to 1,3-PDO via the reductive glycerol dissimilation pathway in C. pasteurianum led to a significant decrease in 1,3-PDO production, to regenerate NAD+ consumed through the pentose phosphate pathway however, glycerol dissimilation persisted with 1,2-PDO formation as an (PPP) (da Cunha and Foster, 1992; Garai-Ibabe et al., 2008). However, alternative redox valve (Pyne, Michael E. et al., 2016). Although the oxidative glycerol dissimilation does not occur naturally under fer- identification of genes encoding putative enzymes related to the1,2- mentative and respiratory conditions due to incomplete and inactive PDO pathway suggests that Clostridium sp. such as C. acetobutylicum, pathways, respectively, in Lactobacillus sp. such that they cannot con- Clostridium difficile, and C. beijerinckii may synthesize 1,2-PDO or its sume glycerol in the absence of a more oxidized carbon source (Kang precursors, this was the first experimental demonstration of 1,2-PDO et al., 2014a, 2014b; Morita et al., 2008; Talarico et al., 1990). Putative production in Clostridium sp. (Huang, K.-x., Rudolph, F.B., Bennett, glycerol kinase (GK) and NADP+-dependent G3P dehydrogenase are G.N., 1999; Liyanage et al., 2001; Pyne et al., 2016a, 2016b). 1,3-PDO common to many Lactobacillus sp., although they are presumably dor- production was recently abolished in C. pasteurianum by deleting the mant or weakly expressed as is the case in certain Clostridium sp. entire dhaBCE operon using Allele-Coupled Exchange (ACE), although (Nölling et al., 2001; Vasconcelos et al., 1994) and K. pneumoniae 1-butanol and side metabolite formation were not significantly affected strains (Forage and Lin, 1982; Wang et al., 2017b). Putative GlyDH was (Schwarz et al., 2017). In contrast, mutation of rex, encoding the redox- identified in the genomes of several Lactobacillus sp., although DHAK is responsive regulator (Rex) that represses expression of genes in the 1- not present, preventing oxidative glycerol dissimilation under fermen- butanol pathway in response to low NADH levels, significantly de- tative conditions (Morita et al., 2008). The reductive pathway of fer- creased 1,3-PDO and butyrate formation with markedly improved 1- mentative glycerol dissimilation begins with the conversion of glycerol butanol production, in spite of reduced glycerol utilization. Finally, to 3-HPA, a process initially thought to occur via either of two distinct mutation of hydA, encoding ferredoxin dehydrogenase (HydA), sig- B12-dependent enzymes, i.e. GDHt and DODHt (Talarico and nificantly increased acetate, lactate, and ethanol formation, with are- Dobrogosz, 1990). However, the three subunits of a putative dehy- duction in butyrate and 1,3-PDO production, and a modest increase in dratase from Lactobacillus reuteri JCM1112 show sequence similarity to the 1-butanol titer (Schwarz et al., 2017). The results are in agreement DODHt of Salmonella typhimurium (i.e. PduCDE encoded by pduCDE) with an earlier study of asRNA-mediated repression of hydA expression (Morita et al., 2008). Moreover, a metabolosome-associated DODHt in C. pasteurianum grown in a complex medium with glucose (Pyne (i.e. PduCDE encoded by pduCDE) was identified in L. reuteri DSM et al., 2015). 20016 that required pre-incubation with 1,2-PDO to achieve high 3-

As DhaB1 of C. butyricum is B12-independent, 1,3-PDO production HPA production from glycerol (Sriramulu et al., 2008). Similar pdu using this organism is of significant interest (Raynaud, and xe, line, Sar, operons have also been found in Lactobacillus collinoides (Sauvageot xe, abal, P., Meynial-Salles, I., Croux, C., Soucaille, P., 2003; Saint- et al., 2002) and L. brevis (Makarova et al., 2006), indicating the Amans et al., 2001). Due to the lack of genetic tools, improving 1,3- common absence of a dedicated GDHt for Lactobacilli. The natural

PDO production in C. butyricum has been limited to derivation of 1,3- ability to synthesize B12 is an attractive feature of many L. reuteri and PDO-tolerant mutants via random mutagenesis (Abbad-Andaloussi certain Lactobacillus panis strains (Kang et al., 2014a; Morita et al., et al., 1995; Reimann et al., 1998; Reimann and Biebl, 1996). The 1,3- 2008; Ricci et al., 2015; Santos et al., 2011; Taranto et al., 2003), al- PDO pathway of C. butyricum was introduced into C. acetobutylicum though it is not common to all Lactobacillus sp. (Pflugl et al., 2014). The (González-Pajuelo et al., 2005) which has more advanced genetic tools reduction of 3-HPA to 1,3-PDO is catalyzed by one or more 1,3- (Pyne et al., 2014). The resulting engineered strains metabolized gly- PDOORs, of which the activity and expression are sensitive to growth cerol anaerobically, resulting in a moderate increase in the 1,3-PDO conditions. L. reuteri DSM 20016 expresses two putative 1,3-PDOORs, titer relative to the natural producer C. butyricum in fedbatch fermen- encoded by lr_0030 and lr_1734, which are active during exponential tations (González-Pajuelo et al., 2005). Improved 1,3-PDO production and stationary growth phases, respectively (Stevens et al., 2011). In from glycerol was achieved in C. beijerinckii via coexpression of gldA addition, lr_0030 is down-regulated upon depletion of glucose, resulting and dhaKLM from E. coli and, compared to the wild-type strain, the in 3-HPA accumulation, a phenomenon also observed in L. panis (Kang engineered strain also had a greatly increased specific growth rate et al., 2013) and L. collinoides (Sauvageot et al., 2000). Among lactate- (Wischral et al., 2016). While the tools available for genetic manip- producing bacteria, L. reuteri has the unique capability to produce and ulation in Clostridium sp. are lagging behind other industrially im- excrete 3-HPA in large quantities. The 3-HPA/1,3-PDO ratio decreases portant bacteria, recent advances such as ACE and CRISPR can accel- as the glucose/glycerol ratio increases in L. reuteri cultures (Luthi-Peng erate the pace of strain development. Notably, 74% of Clostridium sp. et al., 2002), although 1,3-PDO formation is hindered when glycerol is harbor CRISPR-Cas machinery, and native CRISPR-Cas systems can be present in excess (~30 g/L) (Bauer et al., 2010). The latter observation exploited for genome editing with significantly greater editing effi- is not surprising given that expression of dhaT is repressed under high ciency than heterologous platforms (Pyne et al., 2016a, 2016b). Abol- glycerol loading in L. panis (Kang et al., 2013), a process aggravated by ishing 1,3-PDO production in C. pasteurianum did not significantly alter the 3-HPA-mediated inhibition of DhaT (Kang et al., 2014b). Con- metabolite profiles, such that systems level in silico analysis of gene versely, the 1,3-PDO titer increased as the glycerol concentration was mutations combined with an efficient toolkit for genome editing maybe increased up to 70 g/L during batch fermentations of Lactobacillus necessary to dramatically reduce the labor of strain optimization for diolivorans (Pflugl et al., ).2012 high-level 1-butanol production. In the case of C. pasteurianum, high- In general, cell growth can be maintained or enhanced upon the efficiency transformation protocols have been developed (Pyne et al., addition of glycerol to anaerobic glucose cultures of Lactobacillus sp. (da 2013; Schwarz et al., 2017), however, low transformability still remains Cunha and Foster, 1992; El-Ziney et al., 1998; Pflugl et al., 2012) with a an issue for many promising Clostridium sp. (González-Pajuelo et al., metabolic shift from ethanol and lactate to 1,3-PDO and acetate (da 2006; Li et al., 2016a, 2016b) and must be addressed prior to the ap- Cunha and Foster, 1992; El-Ziney et al., 1998; Luthi-Peng et al., 2002; plication of advanced genome engineering technologies for metabolic Pflugl et al., 2012). The formation of 1,3-PDO replaces ethanol and, to a engineering purposes. lesser extent, lactate for NAD+ replenishment, enabling the production of additional ATP via acetate synthesis (da Cunha and Foster, 1992). 3.4. Lactobacillus sp. Lactate can also be oxidized to generate acetate under low sugar concentrations, with 1,3-PDO serving as the major redox outlet (da Cunha 3.4.1. Natural metabolism and Foster, 1992). Production of 3-HP has also been observed in L. Certain Lactobacillus sp. strains are industrially important for lactate reuteri (Ramakrishnan et al., 2015), L. collinoides (Garai-Ibabe et al., production through anaerobic dissimilation of glycerol with sugar co- 2008), and L. diolivorans (Garai-Ibabe et al., 2008), serving to restore

553 A.W. Westbrook et al. Biotechnology Advances 37 (2019) 538–568 the cellular redox balance upon depletion of fermentable sugars (Luo glycerol as the sole carbon source in L. panis (Kang et al., 2014a, et al., 2011a, 2011b; Sriramulu et al., 2008). Expression of pduP from L. 2014b), cofactor engineering via expression of NOX could regenerate reuteri in K. pneumoniae markedly enhanced 3-HP production relative to NAD+ without forming competing side products (i.e. ethanol), and the wild-type strain (Luo et al., 2011a, 2011b), indicating that PduP expression of DhaT from Klebsiella sp. or Clostridium sp. may improve may be associated with the 3-HP pathway in Lactobacillus sp. as is the conversion of 3-HPA to 1,3-PDO while consuming NADH. Moreover, case in Klebsiella sp. (Luo et al., 2012). Strains of L. diolivorans and L. expression of NOX may also alleviate NAD+ depletion in Lactobacillus collinoides isolated from bitter tasting ciders produced 3-HP as a major sp. engineered to overproduce 3-HP. To further enhance glycerol dis- metabolite during fermentation on fructose and glycerol (Garai-Ibabe similation, the native respiratory pathway could be activated and co- et al., 2008). High sugar concentrations favored 1,3-PDO production, expression of glpF from a Gram-positive organism, e.g. a facultative while equimolar quantities of 3-HP and 1,3-PDO were obtained under anaerobe such as B. subtilis, may enhance the functional expression of fructose limitation (Garai-Ibabe et al., 2008). the membrane-bound permease, relative to GlpF from a Gram-negative bacterium. Similarly, an engineered fermentative pathway containing 3.4.2. Strain engineering the native putative GlyDH may facilitate glycerol dissimilation in the Engineering Lactobacillus sp. to enhance glycerol dissimilation has absence of a more reduced carbon source. Finally, in light of the sub- emerged recently for synthesis of 1,3-PDO and 3-HP. An attempt to stantial number of putative LDHs and aldehyde/alcohol dehy- improve 1,3-PDO production in engineered L. reuteri was made through drogenases in Lactobacillus sp., knocking out relevant genes by the expression of yqhD from E. coli, resulting in a significant increase in the CRISPR-Cas9 system could simplify the elimination of lactate and specific 1,3-PDO productivity relative to the wild-type strain ethanol production to enhance target metabolite formation. (Vaidyanathan et al., 2011). However, a shift from acetate and biomass to ethanol and lactate formation was observed, presumably due to the 3.5. Escherichia coli preference of YqhD for NADPH, diminishing the activity of the native NADH-dependent 1,3-PDOOR (i.e. PduQ encoded by pduQ) and, in 3.5.1. Natural metabolism turn, disturbing the natural redox balance (Vaidyanathan et al., 2011). E. coli can naturally dissimilate glycerol aerobically and anaerobi- As part of an assessment of heterologous recombinases in L. reuteri, cally via respiratory and fermentative pathways (Dharmadi et al., mutation of the catabolite repression element (CRE) located upstream 2006). Glycerol is transported into E. coli through facilitated diffusion of the pdu operon resulted in a 3-fold increase in 3-HPA production by GlpF (Richey and Lin, 1972), which can also transport polyols (e.g. when cells grown overnight in a medium containing glucose were erythritol), urea, glycine, and glyceraldehyde (Heller et al., 1980). In transferred to a medium containing glycerol (van Pijkeren et al., 2012). the cytosol, glycerol enters the respiratory or fermentative pathways, The CRE mutant was subsequently evaluated for 3-HP/1,3-PDO co- either of which produce DHAP as an intermediate of glycolysis. In the production in a fedbatch resting cell fermentation and the specific respiratory pathway, glycerol is phosphorylated to G3P by GK (i.e. GlpK productivities of 3-HP and 1,3-PDO increased significantly, relative to encoded by glpK)(Hayashi and Lin, 1967), the presumed limiting step the wild-type strain, with essentially all glycerol being converted to the for aerobic dissimilation of glycerol (Lin, 1976a, 1976b; Zwaig et al., target metabolites (Dishisha et al., 2014). 1970). Subsequently, if oxygen acts as an electron acceptor, G3P is L. panis has been engineered by expressing yqhD from E. coli to converted to DHAP by aerobic-G3PDH (i.e. GlpD encoded by glpD), or enhance 1,3-PDO production from thin stillage with a significant gly- anaerobic-G3PDH (i.e. GlpABC encoded by glpABC) in the presence of cerol content (Kang et al., 2014b). The L. panis strain was further en- electron acceptors other than oxygen (i.e. nitrate and fumarate). In the gineered to dissimilate glycerol as the sole carbon source through me- fermentative pathway, glycerol is oxidized to DHA via NAD+-depen- tabolic introduction of a respiratory pathway (Kang et al., 2014a, dent GlyDH (i.e. GldA encoded by gldA)(Tang et al., 1979), followed by 2014b). However, coexpression of glpK, glpF, glpD, and tpi, encoding phosphorylation of DHA to DHAP via PEP-dependent DHAK (i.e. triosephosphate isomerase (Tpi), from E. coli resulted in poor cell DhaKLM encoded by dhaKLM)(Gonzalez et al., 2008). The expression growth and minimal glycerol consumption with lactate as the major of enzymes in the respiratory and fermentative pathways is under a end metabolite, indicating that NADH generated through the synthetic collective control of the global regulators cAMP-CRP, Cra, ArcA, Fnr, respiratory pathway was not effectively recycled. Accordingly, coex- and RpoS encoded by crp, cra, arcA, fnr, and rpoS, respectively (Iuchi pression of yqhD from E. coli was employed to restore redox poise (Kang and Lin, 1993; Martínez-Gómez et al., 2012). et al., 2014a, 2014b). It was anticipated that YqhD could reduce acetate In the absence an exogenous electron acceptor, E. coli cannot to acetaldehyde, which could be further reduced to ethanol via an en- naturally maintain redox poise through the production of reduced dogenous alcohol dehydrogenase, while simultaneously enhanced 3- compounds other than 1,2-PDO (Dharmadi et al., 2006). The net NADH HPA conversion to 1,3-PDO would occur (Kang et al., 2014a). The cell produced through the lactate, ethanol, succinate, and 1,2-PDO path- density and glycerol utilization increased dramatically upon coexpres- ways are 1, 0, 0, and -1 mol NADH per mol glycerol consumed, re- sion of yqhD, relative to the control strain expressing the synthetic respectively, such that only 1,2-PDO formation can regenerate NAD+. In spiratory pathway, and ethanol and 1,3-PDO were the major reduced addition to redox balance, glycerol fermentation is dependent on pH metabolites. Enhanced 1,3-PDO production was also demonstrated in L. and the availability of CO2 (Durnin et al., 2009). E. coli requires CO2 in - diolivorans by overexpressing a native putative NADPH-dependent 1,3- the form of bicarbonate (HCO3 ) for growth and biosynthesis of various PDOOR (i.e. PDO-DH (NADPH)) (Pflugl et al., 2013). The approach small molecules and cellular components (Dharmadi et al., 2006). resulted in slightly improved cell growth and a moderate increase in Under fermentative conditions, CO2 production mainly relies on the 1,3-PDO formation, indicating that the putative 1,3-PDOOR readily activity of the FHL complex (encoded by fdhF and hycBCDEFG) for the uses NADH or NADPH as a cofactor to convert 3-HPA to 1,3-PDO. conversion of formate to CO2 and H2 (Iuchi and Lin, 1993). Tran- While engineering of glycerol metabolism in Lactobacillus sp. has not scription of the genes encoding the FHL complex requires formate and been studied extensively, many of these microorganisms have been an acidic pH (Sawers et al., 2004). An alkaline environment causes an - granted the Generally Recognized as Safe (GRAS) status from the FDA, equilibrium shift toward HCO3 , which cannot be transported across the USA, making them ideal hosts for the conversion of glycerol to value- cellular membrane of E. coli, thus reducing CO2 availability inside the added products on an industrial scale. In particular, further exploration cell (Dharmadi et al., 2006). H2 is co-produced with CO2 and can act as of 1,3-PDO production in Lactobacillus sp. is warranted due to their an electron donor, further contributing to the redox imbalance during natural production potential and apparent minimal 2,3-BDO formation glycerol fermentation and potentially impairing growth (Dharmadi as a side metabolite. For example, to overcome the NADH accumulation et al., 2006). Other glycerol fermentation products of E. coli include issue upon the introduction of a respiratory pathway for dissimilation of ethanol, succinate, and acetate (Gonzalez et al., 2008), and ethanol

554 ..Wsboke al. et Westbrook A.W. Table 3 Summary of literature for E. coli strains engineered for enhanced natural metabolite production from glycerol.

Parent strain Product Genetic strategy Titer (g/L) Cultivation mode Reference

ATCC8739 ethanol -Mutate frdABCD, ackA-pta and poxB, and ldhA to reduce succinate, acetate, and lactate formation, respectively, and mutate gldA 47 Fedbatch (Wong et al., 2014) and glpK for controlled expression -Overexpress dhaKLM, gldA, glpK, and adhE for increased carbon flux and ethanol formation LY180 ethanol Fosmid containing metagenomic fragment isolated from anaerobic reactor 75 Batch (Loaces et al., 2016) MG1655 ethanol -Mutate frdA, pta, and fdhF to reduce succinate, acetate, and CO2, and H2 formation, respectively 10 Batch (Yazdani and Gonzalez, 2008) -Overexpress gldA and dhaKLM for increased carbon flux through the fermentative branch BW25113 H2 -Mutate hycA, hyaB, hybC, hycE, hyfG, pflB, fhlA, fdhF, gldA, or glpA to affect2 H formation N/A Crimp-top vials (Sanchez-Torres et al., 2013) BW25113 H2 Mutate aroM, gatZ, ycgR, or yfgI to enhance H2 formation See reference Crimp-top vials (Tran et al., 2015) BW25113 H2 Mutate frdC, ldhA, fdnG, ppc, narG, mgsA, and hycA to enhance H2 formation 158 μmol/mg Crimp-top vials (Tran et al., 2014) protein ATCC8739 succinate -Mutate pflB to reduce formate, ethanol, and acetate formation 12 Flask (Zhang et al., 2010) -Mutate ptsI to shift PEP flux away from DHAK -Mutation in promotor region of pck gene to increase expression level MG1655 succinate -Mutate adhE, pta and poxB, and ldhA to reduce ethanol, acetate, and lactate formation, respectively 14 Flask (Blankschien et al., 2010) -Mutate ppc to remove selection requirement -Express pyc (L. lactis) for succinate production from pyruvate node BL21 (DE3) succinate -Mutate ppc and chromosomal expression of glyoxylate shunt genes aceBAK followed by directed evolution for increased glyoxylate 43 Fedbatch (Li et al., 2013) shunt activity and reactivation of ppc -Mutate sdhCDAB to reduce product degradation -Chromosomal expression of sucAB to reduce α-ketoglutarate formation MG1655 D-lactate -Mutate pta, adhE, and frdA to reduce acetate, ethanol, and succinate formation, respectively 45 Flask (Mazumdar et al., 2010) -Mutate dld to reduce D-lactate degradation -Overexpress glpD and glpK to increase carbon flux through the respiratory pathway BL21 (DE3) D-lactate -Mutate pta, adhE, frdA, pflB, and pflCD to reduce side metabolite formation –Mutate mgsA and dld to reduce MG formation and D- 105 Fedbatch (Wang et al., 2015a, 2015b) 555 lactate degradation, respectively -Directed evolution for increased crude glycerol utilization -Chromosomal expression of glpD and glpK to increase carbon flux through the respiratory pathway, and chromosomal expression of focA and ldh (L. helveticus) to increase lactate export and formation, respectively MG1655 L-lactate -Mutate pflB, pta, adhE, and frdA to reduce side metabolite formation 50 Flask (Mazumdar et al., 2013) -Mutate mgsA and ldhA to prevent D-lactate formation, and mutate lldD to reduce L-lactate degradation -Overexpress glpK and glpD to increase carbon flux through the respiratory pathway -Chromosomal expression of ldh (S. bovis) for L-lactate formation BW 25113 L-lactate -Mutate ackA, pta, pps, pflB, poxB, adhE, and frdA to reduce side metabolite formation 142 Fedbatch (Tian et al., 2013) -Mutate ldhA and lld to prevent D-lactate formation and product degradation, respectively -Chromosomal expression of ldh (B. coagulans) for L-lactate formation W3110 L-phenyl -Mutate tyrA to reduce L-tryptophan formation 13 Fedbatch (Weiner et al., 2014) alanine -Mutate pykA and pykF to increase available PEP, and mutatepheA and aroF for controlled expression -Overexpress aroFBL and feedback resistant pheAfbr for increased shikimate and L-phenylalanine formation, respectively -Feed lactate and ammonia BL21 (DE3) fumarate -Previously evolved succinate producing strain containing ppc mutation and expressing chromosomal aceBAK 42 Fedbatch (Li et al., 2014)

-Mutate fumABC and aspA to reduce fumarate degradation Biotechnology Advances37(2019)538–568 -Overexpress ppc to increase fumarate synthesis MG1655 formate -Mutate frdA, pta, and fdhF to reduce side metabolite formation 7 Batch (Yazdani and Gonzalez, 2008) -Overexpress gldA and dhaKLM for increased glycerol utilization MG1655 FFAs -Mutate fadD and ptsG to enhance FFA formation, and mutate fabR to remove repression of fatty acid synthesis genes 5 Flask (Wu et al., 2014) -Overexpress nadK and pntAB to increase intracellular redox equivalents, and express an acyl-ACP thioesterase (R. communis) for increased FFA formation BL21 (DE3) DHA -Coexpress nox (E. faecalis) and gldA for increased NAD+ regeneration and DHA formation, respectively 2 Test tube (Yang et al., 2013) -Exogenous supply of NAD+ Lin43 acetol -Mutate gloA to reduce MG degradation 5 Flask (Zhu et al., 2013) -Express yqhD for increased acetol synthesis (continued on next page) A.W. Westbrook et al. Biotechnology Advances 37 (2019) 538–568 , 2 formation is essential as it generates ATP and is redox-balanced (Murarka et al., 2008). Although succinate production is also redox- balanced, it contributes minimally to ATP generation (Unden and

fdnG , formate Kleefeld, 2004), making it non-essential during glycerol fermentation glpA , anaerobic (Murarka et al., 2008), whereas acetate production is often marginal during glycerol fermentation. poxB , pyruvate dehy-

pykF , pyruvate kinase I; The highly reduced nature of glycerol makes it an ideal feedstock for narG , respiratory nitrate -arabino-heptulosonate-7-

D the production of reduced compounds and derived value-added pro- hycE , formate hydrogenlyase

( Clomburg and Gonzalez, 2011 ) ducts. Anaerobic conditions favor the production of reduced compounds as oxygen is not available as an electron acceptor. However, anaerobic glycerol fermentations require supplementation of rich frdABCD , fumarate reductase enzyme media components to counteract the production of reducing equiva- κ κ

aroFBL , 3-deoxy- lents generated via biomass formation ( glycerol=4.7; biomass=4.3) and pntAB , protein PntAB; nadK , NAD kinase; fdhf , formate dehydrogenase H; gloA , lactoylglutathione lyase; the low activity of the 1,2-PDO pathway (Durnin et al., 2009). In pykA , pyruvate kinase II; Cultivation mode Reference Batch

yqhD , NAD(P)H-dependent 1,3-propanediol dehy- contrast to anaerobic fermentation, microaerobic conditions introduce limiting amounts of oxygen, effectively acting as an electron acceptor for redox equivalents generated from biomass, such that rich media components are no longer required while the production of reduced pheA , P-protein; compounds is maintained (Durnin et al., 2009).

3.5.2. Strain engineering Titer (g/L) 6 Table 3 and Table 4 summarize strategies for strain engineering of E. pyc , pyruvate carboxylase;

mgsA , methylglyoxal synthase; coli for enhanced production of natural and non-native metabolites gldA , glycerol dehydrogenase; dhaKLM , DHA kinase, PEP-dependent dihydroxyacetone kinase ADP-binding hycA , formate hydrogenlyase regulatory protein; from glycerol, respectively. Selective target products are used for yfgI , uncharacterized protein; technical illustration in this section. adhE , aldehyde-alcohol dehydrogenase; fadD , long-chain fatty acid CoA ligase;

3.5.2.1. 1,3–PDO. Preliminary attempts to produce 1,3-PDO in E. coli by expressing the dha regulon from either K. pneumoniae (Sprenger et al., 1989; Tong et al., 1991) or C. freundii (Daniel and Gottschalk,

frdA , fumarate reductase flavoprotein subunit (anaerobic); 1992) resulted in substantially lower 1,3-PDO titers than those obtained , carbon dioxide; DHA, dihydroxyacetone; DHAK, dihydroxyacetone kinase, FFA, free fatty acids; H 2 with natural production hosts (Cameron et al., 1998; Chotani et al., ptsI , PEP phosphotransferase; 2000). As a result, more advanced strain engineering strategies were

lldD , L-lactate dehydrogenase; developed to enhance 1,3-PDO production in E. coli, primarily ycgR , flagellar brake protein; aspA ; aspartate ammonia-lyase; pflCD , pyruvate formate lyase subunits C and D;

hybC , hydrogenase-2 large chain; including: (1) the improved expression of genes involved in the reductive branch of glycerol dissimilation from natural 1,3-PDO producers; (2) the expression of native putative NADPH-dependent 1,3-PDOOR, YqhD; and (3) reducing the formation of the toxic metabolites MG, G3P, and 3-HPA. MG is an intermediate of 1,2-PDO fabR , HTH-type transcriptional repressor; tyrA , T-protein; focA , formate transporter 1; production, and concentrations exceeding 0.5 mM can inhibit cell gatZ , D-tagatose-1,6-bisphosphate aldolase subunit;

aroM , protein AroM; growth (Ackerman et al., 1974; Booth et al., 2003; MacLean et al., 1998a, 1998b). Anaerobic fermentation of glycerol by E. coli strains expressing the dha regulon from K. pneumoniae resulted in the ldhA , lactate dehydrogenase A; accumulation of MG to toxic levels with lowered 1,3-PDO production

pflB , formate acetyltransferase 1; (Zhu et al., 2001). As MG can be converted to S- lactoylglutathinone by hyaB , hydrogenase-1 large chain; glyoxalase I, and subsequently to lactate by glyoxalase II (MacLean et al., 1998a, 1998b), expression of glyoxalase I, i.e. Glo1 (encoded by glo1 from Pseudomonas putida), reduced intracellular MG levels and ptsG , PTS system glucose-specific EIICB component; moderately enhanced 1,3-PDO production (Zhu et al., 2001). Similarly, ( C. freundii ) for ATP dependent functionality (compared to native PEP dependence) to reduce acetate and lactate formation, respectively

for increased 1,2-PDO formation both cell growth and 1,3-PDO production were inhibited when the ldhA

dhaKL intracellular G3P concentration exceeded 10 mM in E. coli (Lin, 1976a, yqhD

glpK , glycerol kinase; 1976b; Zhu et al., 2002), and the addition of an exogenous electron ldh , lactate dehydrogenase; pck , PEP carboxykinase; fumABC , fumarase subunits A, B, and C; sucAB , alpha-ketoglutarate dehydrogenase; acceptor such as fumarate could increase the activity of GlpABC and, in pta , and turn, curb G3P accumulation and dramatically improve 1,3-PDO gldA , and and express and dld , quinone-dependent D-lactate dehydrogenase; production (Zhu et al., 2002). aceBAK , isocitrate lyase, malate synthase, and isocitrate dehydrogenase kinase/phosphorylase; msgA ,

ackA dhaK The reductive branch of glycerol dissimilation from K. pneumoniae, fhlA , formate hydrogenlyase transcriptional activator; pta , phosphotransacetylase; C. freundii, and C. butyricum have been introduced in E. coli to facilitate 1,3-PDO production. While dhaB123 and dhaT are divergently ex- -Mutate -Express

nox , NADH oxidase; pressed from individual promoters in K. pneumoniae, this arrangement resulted in a lower 1,3-PDO titer in E. coli, compared to the coexpression of dhaB123 and dhaT from a single promoter (Skraly et al., 1998).

ackA , acetate kinase; Furthermore, native YqhD could convert 3-HPA to 1,3-PDO more efficiently than DhaT from K. pneumoniae (Emptage et al., 2003b), and the ppc , PEP carboxylase; hyfG , hydrogenase-4 component G;

frdC , fumarate reductase subunit C; preference of YqhD for NADPH may have contributed to higher 1,3- ( continued ) PDO titers by altering the reduced/oxidized cofactor ratios (Nakamura and Whited, 2003). Coexpression of gdrAB and dhaB123 from K. Parent strain Product Genetic strategy MG1655 1,2-PDO -Mutate hydrogen gas; MG, methylglyoxal; NAD+, nicotinamide adenine dinucleotide; PEP, phosphoenolpyruvate. phosphate synthase isoenzyme, 3-dehydroquinate synthase, and shikimatedehydrogenase kinase; N subunit alpha; drogenase; sdhCDAB , succinate dehydrogenase complex; subunit, and protein-lysine deacetylase; complex; glycerol-3-phosphate dehydrogenase subunit A; subunit 5; reductase 1 alpha chain; drogenase. Abbreviations: 1,2-PDO, 1,2-propanediol; acyl-ACP, acyl carrier protein; ATP, adenosine triphosphate; CO Table 3 Gene products: pneumoniae with native yqhD significantly increased 1,3-PDO

556 A.W. Westbrook et al. Biotechnology Advances 37 (2019) 538–568

Table 4 Summary of literature for E. coli strains engineered to produce 1,3-PDO and 3-HP from glycerol.

Parent strain Product Genetic strategy Titer (g/ Cultivation mode Reference L)

AG1 1,3-PDO Express dha regulon (K. pneumoniae) N/A Flask (Tong et al., 1991) ELC707 1,3-PDO Express dha regulon (C. freundii) 5 Flask (Daniel and Gottschalk, 1992) AG1 1,3-PDO Coexpress dhaB and dhaT (K. pneumoniae) from trc promoter 6 Fedbatch (Skraly et al., 1998) AG1 1,3-PDO -Express dha regulon (K. pneumoniae) 2 Flask (Zhu et al., 2001) -Express glo1 (P. putida) to reduce MG formation AG1 1,3-PDO -Express dha regulon (K. pneumoniae) 4 Flask (Zhu et al., 2002) -Mutate glpK and feed fumarate to reduce G3P formation JM109 1,3-PDO -Coexpress dhaB and gdrAB (K. pneumoniae), and yqhD 13 Fedbatch (Hong et al., 2015) -Feed succinate to decrease NAD+/NADH ratio JM109 1,3-PDO Coexpress dhaBCE (C. freundii) and yqhD 41 Fedbatch (Zhang et al., 2006a, 2006b) BL21 1,3-PDO Coexpress dhaBCEFG (C. freundii) and dhaT (K. pneumoniae) 11 Fedbatch (Przystałowska et al., 2015) K-12 ER2925 1,3-PDO Coexpress dhaB12 (C. butyricum) and yqhD 104 Fedbatch (Tang et al., 2009) JM109 1,3-PDO -Coexpress yqhD, and dhaB and gdrAB (K. pneumoniae) 14 Flask (Yang et al., 2018) -Coexpress gapN (C. acetobutylicum) to generate NADPH, and fine-tune expression via 5’-UTR engineering -mutate ptsG and overexpress galP and glk to relieve catabolite repression during glycerol/glucose co-feeding BL21 3-HP Coexpress dhaB123 (K. pneumoniae) and aldH 0.6 Fedbatch (Raj et al., 2008) BL21 3-HP Coexpress dhaB123 (K. pneumoniae) and aldH from low-copy and high-copy 31 (Raj et al., 2009) plasmids, respectively BL21 3-HP Coexpress dhaB and dhaR (L. brevis) and aldH 14 Flask (Kwak et al., 2013) W3110 3-HP -Coexpress dhaB123 and gdrAB from K. pneumoniae, and aldH 42 Fedbatch (Jung et al., 2014) -Overexpress glpF to increase glycerol uptake -Mutate ackA-pta, yqhD, and glpR to reduce acetate, 3-HP, and G3P formation, respectively BL21 3-HP -Coexpress dhaB and dhaR (L. brevis), and PSALDH (P. aeruginosa) 57 Fedbatch (Kim et al., 2014) -Mutate glpK and yqhD to reduce G3P and 3-HP formation, respectively W 3-HP Coexpress dhaB and gdrAB (K. pneumoniae), and KGSADH (A. brasilense) 42 Fedbatch (Sankaranarayanan et al., 2014) W3110 3-HP -Coexpress dhaB and gdrAB (K. pneumoniae), and engineered gabD4 (C. necator) 72 Fedbatch (Chu et al., 2015) with point mutations E209Q and E269Q -Mutate pta-ackA and yqhD to reduce acetate and 3-HP formation, respectively BW25113 3-HP -Implement metabolic toggle switch to conditionally repress the expression of 6 Fedbatch (Tsuruno et al., 2015) gapA in ΔgapA -Coexpress dhaB123 and gdrAB (K. pneumoniae), and araE (A. brasilense) -Mutate yqhD and to reduce 1,3-PDO formation B (ATCC11303) 3-HP Coexpress dhaB123 and gdrAB (K. pneumoniae), KGSADH (A. brasilense), and 5 Fedbatch (Honjo et al., 2015) pduPLW (K. pneumoniae)

Gene products: ackA, acetate kinase; aldH, aldehyde dehydrogenase; araE, α-ketoglutaric semialdehyde dehydrogenase; dhaB123, glycerol dehydratase; dhaBCDEFG, glycerol dehydratase and reactivation factors; dhaBT, glycerol dehydratase and 1,3-propanediol oxidoreductase; dhaR, dihydroxyacetone reductase; dhaT, 1,3- propanediol oxidoreductase; gabD4, aldehyde dehydrogenase; gapA, glyceraldehyde 3-phosphate dehydrogenase; gdrAB, glycerol dehydratase reactivase; glo1, glyoxylase 1; glpF, glycerol uptake facilitator protein; glpK, glycerol kinase; glpR, glycerol-3-phosphate regulon repressor; KGSADH, alpha-ketoglutarate semialdehyde dehydrogenase; pduPLW, aldehyde dehydrogenase, phosphate propanoyltransferase, and propionate kinase; PSALDH, semialdehyde dehydrogenase complex; pta, phosphotransacetylase; yqhD, NAD(P)H-dependent 1,3-propanediol dehydrogenase. Abbreviations: 3-HP, 3-hydroxypropionate; G3P, glycerol-3-phosphate; MG, methylglyoxal; NAD+, nicotinamide adenine dinucleotide.

production relative to the strain expressing only dhaB123 and yqhD, (Zhang et al., 2006a, 2006b). On the other hand, a considerable amount (Hong et al., 2015), indicating that glycerol- and/or oxygen-mediated of acetate was generated by engineered E. coli expressing dhaBCE and inactivation of DhaB was a critical issue in these E. coli strains. Ad- dhaFG from C. freundii and dhaT from K. pneumoniae, leading to cell ditionally, supplementation of the TCA cycle intermediates succinate, growth inhibition and decreased 1,3-PDO production (Przystałowska fumarate, and malate significantly reduced the NAD+/NADH ratio and et al., 2015). Finally, coexpression of native yqhD with dhaB1 and further improved 1,3-PDO production (Hong et al., 2015). Subse- dhaB2 from C. butyricum resulted in high-level 1,3-PDO production with quently, gapN, encoding NADP+-dependent glyceraldehyde-3-phos- the titer reaching 104 g/L in an anaerobic fedbatch fermentation, al- phate dehydrogenase (GAPDN) from C. acetobutylicum, was coexpressed though acetate accumulation eventually limited culture performance with native yqhD, and dhaB123 and gdrAB from K. pneumoniae, to re- (Tang et al., 2009). This strain engineering approach is particularly generate NADPH, resulting in significantly improved 1,3-PDO produc- attractive, as the expression of DhaB1 from C. butyricum does not re- tion relative to the strain coexpressing yqhD, dhaB123, and gdrAB (Yang quire B12. et al., 2018). Fine-tuning the expression of gapN via modification of its Although large-scale 1,3-PDO production using engineered E. coli 5’-UTR led to significant improvements to culture performance, which has been demonstrated (Celińska, 2010), strategies to eliminate bio- was further enhanced by mutating ptsG, encoding the phospho- processing limitations and further enhance culture performance require transferase system (PTS) glucose-specific EIICB protein, and over- to be developed. For example, complete growth inhibition of E. coli expressing galP (encoding galactose permease; GalP) and glk (encoding occurred for 1,3-PDO concentrations exceeding 120 g/L (Cameron glucokinase; GLK) to relieve carbon catabolite repression when glycerol et al., 1998), such that improving the 1,3-PDO tolerance of E. coli and glucose were provided as co-substrates (Yang et al., 2018). Relative through directed evolution or rational strain engineering may be an to the expression of genes from the reductive branch of glycerol dis- effective strategy to improve 1,3-PDO production. Cell membrane en- similation from K. pneumoniae, coexpression of native yqhD and dhaBCE gineering was applied to improve rates of biocatalysis in E. coli (Chen, from C. freundii resulted in significantly improved 1,3-PDO production 2007), protein secretion (Cao et al., 2017) and biopolymer production

557 A.W. Westbrook et al. Biotechnology Advances 37 (2019) 538–568

(Westbrook et al., 2018) in B. subtilis, and solvent and acid tolerance in inactivation to enhance 3-HP production from glycerol (Tokuyama C. acetobutylicum (Zhao et al., 2003) and E. coli (Tan et al., 2017), and, et al., 2014). Mutation of tpiA in a strain coexpressing dhaB123 and hence, may be an effective strategy to enhance the 1,3-PDO tolerance of gdrAB from K. pneumoniae and native aldH increased the 3-HP titer by E. coli. To restore the potential cofactor imbalance arising from the 2-fold, while mutation of zwf had only a minor effect on culture per- conversion of 3-HPA to 1,3-PDO by YqhD, native zwf, encoding formance. Moreover, mutation of yqhD significantly reduced 1,3-PDO NADP+-dependent glucose 6-phosphate-1-dehydrogenase (Zwf), can be formation and, in turn, 3-HP production was markedly increased, overexpressed to regenerate NADPH with low-level glucose supple- compared to the ΔtpiA Δzwf double mutant coexpressing dhaB123, mentation. Moreover, the accumulation of acetate to inhibitory levels gdrAB, and aldH (Tokuyama et al., 2014). Similarly, mutation of ackA- observed during high-level 1,3-PDO production (Tang et al., 2009) may pta and yqhD significantly reduced acetate and 1,3-PDO formation, with be relieved by mutation of ptsH, encoding the histidine protein of the a concomitant increase in 3-HP production in a strain coexpressing sugar PTS that participates in the phosphorylation of DHA by DhaKLM, dhaB123 and gdrAB from K. pneumoniae and native aldH (Chu et al., which significantly reduced acetate formation during anaerobic succi- 2015). Moreover, 3-HP production was further enhanced by expressing nate production from glycerol (Zhang et al., 2010). gabD4, encoding ALDH from C. necator (GabD4), in place of native aldH, while expression of engineered GabD4, with point mutations E209Q 3.5.2.2. 3-HP. Strain engineering strategies to enhance 3-HP and E269Q, significantly improved 3-HP production during a fedbatch production in E. coli have primarily focused on balancing the co-cultivation using glycerol and glucose (Chu et al., 2015). Finally, activities of GDHt and ALDH in the synthetic 3-HP pathway (Fig. 1), repressing the expression of genes in central metabolic pathways can and limiting side metabolite formation. Low-level 3-HP production was redirect carbon flux toward target metabolite production without first demonstrated in E. coli via coexpression of DhaB from K. compromising host cell viability. A previously engineered metabolic pneumoniae with one of four ALDHs, of which AldH4 from S. toggle switch (Gardner et al., 2000) was used to conditionally repress cerevisiae, encoded by aldH4, was most effective for converting 3-HPA the expression of gapA, encoding glyceraldehyde 3-phosphate dehy- to 3-HP (Suthers and Cameron, 2005). Excessive 3-HPA accumulation drogenase (GapA), in a ΔgapA mutant coexpressing dhaB123 and gdrAB was observed to hinder 3-HP production in a strain coexpressing from K. pneumoniae and araE from A. brasilense to redirect carbon flux dhaB123 from K. pneumoniae and native aldH (encoding AldH) from from glycolysis to the synthetic 3-HP pathway, while minimizing individual low-copy plasmids, due to an imbalance between the growth inhibition observed in ΔgapA mutants (Tsuruno et al., 2015). activities of DhaB and AldH (Raj et al., 2008). Subsequent work The conditional repression of gapA expression resulted in a significant revealed that the activity of GdrAB was critical to prevent the increase in 3-HP production compared to the strain coexpressing inactivation of DhaB, and the coexpression of native aldH, and dhaB123, gdrAB, and araE, whereas mutation of yqhD dramatically re- dhaB123 and gdrAB from K. pneumoniae improved the 3-HP titer by duced 1,3-PDO formation and, in turn, improved the 3-HP titer and 6-fold (Rathnasingh et al., 2009) relative to the corresponding strain yield (Tsuruno et al., 2015). without gdrAB coexpression (Raj et al., 2008). Moreover, the expression Other technical issues to be addressed for enhanced 3-HP produc- of KGSADH from A. brasilense, in place of native aldH, further increased tion from glycerol in E. coli include recycling of NADH and cir- the 3-HP titer by 2.5-fold (Rathnasingh et al., 2009). Fine-tuning the cumventing the requirement of B12-dependent GDHt. Excess NADH may expression of dhaB123 from K. pneumonia by engineering its 5’- UTR accumulate during 3-HP production, potentially resulting in the for- also served to manage 3-HPA accumulation (Lim et al., 2016). KGSADH mation of reduced side metabolites. While NADH can be oxidized by from A. brasilense was expressed from a relatively strong promoter in a NOX using molecular oxygen without diverting carbon from 3-HP highly 3-HPA-tolerant E. coli strain that also expressed dhaB123 with production, B12-independent DhaB1 from C. butyricum may not function engineered 5’-UTRs from a separate promoter, resulting in a substantial in the presence of oxygen (Andreeßen et al., 2010; Tang et al., 2009), increase in the 3-HP yield (Lim et al., 2016). Lastly, parallel expression such that a NAD+ regeneration system functional under anaerobic of the partial PDU pathway (i.e. pduPLW) from K. pneumoniae and araE, conditions would be required in this context. NADH:quinone oxidor- encoding α-ketoglutaric semialdehyde dehydrogenase from A. eductase II, encoded by ndh in E. coli, oxidizes NADH during nitrate brasilense (AraE), in a strain also expressing dhaB123 and gdrAB from respiration (Tran et al., 1997), such that coexpression of native ndh, and K. pneumoniae resulted in a significantly increased 3-HP titer, compared dhaB1 and dhaB2 from C. butyricum (in addition to ALDH) may facil- to a strain that did not express pduPLW, suggesting that conversion of 3- itate anaerobic production of 3-HP without B12 if exogenous nitrate is HPA to 1,3-PDO may have decreased due to increased 3-HPA flux provided. Alternatively, protein engineering of DhaB1 to reduce its through the partial PDU pathway (Honjo et al., 2015). sensitivity to oxygen may enable B12-independent 3-HP production Several strategies were explored to minimize the formation of major under microaerobic conditions. A similar strategy was employed for side metabolites, including acetate, lactate, succinate, and 1,3-PDO, for aerobic 1-propanol production through the L-threonine degradation enhanced 3-HP production in engineered E. coli. Inactivating ackA-pta pathway, whereby engineered aero-tolerant AdhE successively con- and yqhD in a strain coexpressing dhaB123 and gdrAB from K. pneu- verted propionyl-CoA to propionaldehyde and 1-propanol (Choi et al., moniae and native aldH, significantly decreased acetate and 1,3-PDO 2012). In the absence of B12 dependency, E. coli ΔyqhD mutants are production (Jung et al., 2014). 3-HP production increased by 2-fold in superior hosts for 3-HP production relative to other microbial hosts the ΔackA Δpta ΔyqhD triple mutant compared to the parent strain discussed herein due to the minimal formation of 1,3-PDO. coexpressing dhaB123, gdrAB, and aldH, while mutation of glpR, encoding the repressor of the glp regulon (GlpR), and overexpression of 3.5.2.3. Bio(co)polymers. PHAs are a family of biologically produced glpF modestly enhanced 3-HP production (Jung et al., 2014). On the polyesters composed of various (R)-hydroxycarboxylates as the other hand, inactivating respiratory glycerol dissimilation via mutation monomers (Mozzi et al., 2006; Teeka et al., 2012) and have emerged of glpK significantly improved 3-HP production in a strain coexpressing as substitutes for conventional plastics due to their thermoplastic and dhaB and dhaR, encoding the reactivation factor of DhaB (DhaR), from elastomer properties, biodegradability, non-toxicity, and structural L. brevis, and native aldH (Kim et al., 2014; Kwak et al., 2013). More- diversity (Leong et al., 2014; Reddy et al., 2003; Teeka et al., 2012). over, mutation of yqhD significantly improved 3-HP production in the Unlike most PHAs, P(3HP) is not naturally produced by bacteria ΔglpK mutant, and the expression of PSALDH, encoding semialdehyde (Linares-Pastén et al., 2015), leading to the exploration of synthetic dehydrogenase from Pseudomonas aeruginosa, in place of aldH, further pathways in engineered hosts such as E. coli. Marginal P(3HP) enhanced culture performance (Kim et al., 2014). In silico analysis of production was achieved in E. coli coexpressing dhaB1 from C. gene mutations based on a genome-scale metabolic model identified butyricum, pduP from Salmonella enterica, and phaC from C. necator, tpiA, encoding triosephosphate isomerase (TpiA), and zwf as targets for from either pure or crude glycerol (Andreeßen et al., 2010). On the

558 A.W. Westbrook et al. Biotechnology Advances 37 (2019) 538–568 other hand, coexpressing dhaB123 and gdrAB from K. pneumoniae with expression of GDHt are technical challenges to overcome for enhanced pduP from S. typhimurium and phaC from C. necator resulted in a high biopolymer production in E. coli. Excess NADH can accumulate during P specific P(3HP) yield using glucose and glycerol as co-substrates ina (3HP) production due to the conversion of 3-HPA to 3-HP-CoA via fedbatch cultivation (Wang et al., 2013a, 2013b). In spite of the high PduP, potentially hindering culture performance (Andreeßen et al., specific P(3HP) yield, plasmid instability was an issue thatwas 2010), while NADPH is consumed through the conversion of β-keto- addressed using a plasmid addiction system for the coexpression of valeryl-CoA to 3-HV-CoA by PhaB during P(3HB-co-3HV) production. pduP from S. typhimurium and phaC from C. necator, and chromosomal Accordingly, the same strategies proposed to recycle reducing equiva- expression of dhaB123 and gdrAB from K. pneumoniae, resulting in a lents during B12-independent 3-HP and 1,3-PDO production may be high specific P(3HP) yield of 68% of dry cell weight (dcw) in afedbatch effective for P(3HP) and P(3HB-co-3HV) production, respectively. cultivation (Gao et al., 2014). To circumvent the requirement of B12- Protein engineering is also a potential strategy to alter bio(co)polymer dependent GDHt, P(3HP) was produced via the β-alanine pathway in E. composition. Expression of engineered PhaJ from A. hydrophila con- coli (Wang et al., 2014a, 2014b, 2014c). In this engineered redox- taining a V130A point mutation substantially increased the 3-hydro- neutral pathway, native L-aspartate decarboxylase (i.e. PanD encoded xyhexanoate (3-HH) fraction in P(3HB-co-3HH) produced in E. coli, by panD) generated β-alanine that was converted to P(3HP) through without compromising the specific P(3HB-co-3HH) yield (Hu et al., subsequent actions of a β-alanine-pyruvate transaminase from P. putida 2007). Moreover, bio(co)polymer content can be modified by manip- (i.e. GabT encoded by gabT), native 3-hydroxyacid dehydrogenase (i.e. ulating glycerol dissimilation through either the fermentative or re- YdfG encoded by ydfG) and PrpE, and PhaC from C. necator. Although spiratory pathways as previously noted (Srirangan et al., 2016b). exogenous B12 was not required, P(3HP) production was low (Wang et al., 2014a, 2014b, 2014c), with only a modest improvement obtained 3.5.2.4. Other products. Production of 1-propanol from glycerol in E. by expressing panD from Corynebacterium glutamicum in place of native coli was demonstrated using two distinct pathways. In the first panD (Lacmata et al., 2017). Lastly, engineered E. coli was used in a approach, the natural 1,2-PDO pathway of E. coli was modified using two-stage cultivation with resting cells of L. reuteri for P(3HP) an ATP-dependent DHAK, encoded by dhaK from S. blattae, and production from glycerol (Linares-Pastén et al., 2015). Due to a high extended using components of the pdu operon from K. pneumoniae 3-HPA tolerance, L. reuteri was used in the first stage to convert glycerol (Matsubara et al., 2016). 1,2-PDO was converted to propionaldehyde to 3-HPA, which was, in turn, used as a substrate by E. coli coexpressing via PduCDE with the reactivation factor PduGH, and then to 1-propanol pduP from L. reuteri (Sabet-Azad et al., 2013) and phaC from via PduQ. In contrast to native PEP-dependent DhaKLM, ATP- Chromobacterium sp. (Bhubalan et al., 2011), leading to a high dependent DHAK decreased reliance on pyruvate production and, in specific P(3HP) yield (Linares-Pastén et al., 2015). Although not turn, increased flux into the 1-propanol pathway (Matsubara et al., directly related to engineering of glycerol metabolism, other studies 2016). The second strategy to achieve 1-propanol production from have shown the potential of using pure or crude glycerol for the glycerol involved the activation of the dormant Sbm operon of E. coli production of poly(3-hydroxybutyrate) (de Almeida et al., 2007; (Srirangan et al., 2013; Srirangan et al., 2014). This pathway begins Ganesh et al., 2015; Shah et al., 2014; Shalel Levanon et al., 2005). with the conversion of succinyl-CoA to L-methylmalonyl-CoA via a Due to high crystallinity, homopolymer PHAs are too brittle and stiff methylmalonyl-CoA mutase (Sbm encoded by sbm), followed by the for many industrial applications (Leong et al., 2014), and such technical formation of propionyl-CoA from succinyl-CoA by a methylmalonyl- drawbacks can be overcome by the use of copolymers (Singh and CoA decarboxylase (YqfG encoded by yqfG), with a propionyl- Mohanty, 2007). P(3HB-co-3HV) was produced in engineered E. coli CoA::succinate transferase (YgfH encoded by ygfH) facilitating the with a high specific yield of 80% of dcw via coexpression of bktB, en- interconversion between succinyl-CoA and propionyl-CoA. The coding β-ketothiolase (BktB), phaB, encoding acetoacetyl-CoA reactivation of the Sbm operon introduced an intracellular carbon flux ductase (PhaB), and phaC, from R. eutropha, in addition to native sucCD, competition between the C2-fermentative pathway (with ethanol and using glycerol as the primary carbon source with supplemental succi- acetate as the major metabolites) and C3-fermentative pathway (with 1- nate and propionate (Bhatia et al., 2015). Previous studies have re- propanol and propionate as the major metabolites), and glycerol was ported high C3 metabolite production from glycerol due to more identified as a suitable carbon source favoring direction ofthe NADPH and ATP generation relative to glucose, favoring threonine dissimilated carbon flux toward the C3-fermentative pathway biosynthesis and directing carbon flux toward propionyl-CoA, a pre- (Srirangan et al., 2014). The propionyl-CoA derived from the Sbm cursor to (R)-3-hydroxyvaleryl-CoA [(R)-3-HV-CoA] (Tseng et al., operon and acetyl-CoA were fused by either PhaA or BktB from C. 2010). High-level propionyl-CoA and, in turn, 1-propanol production necator to form 3-ketovaleryl-CoA, which was further converted to was achieved from glycerol without supplemental succinate and pro- butanone via a ketone-formation pathway containing acetoacetyl- pionate by activating the native genomic sleeping beauty mutase (Sbm) CoA:acetate/butyrate:CoA transferase (i.e. CtfAB encoded by ctfAB) operon (Srirangan et al., 2013; Srirangan et al., 2014). This approach and acetoacetate decarboxylase (i.e. Adc encoded by adc) from C. was extended to P(3HB-co-3HV) production by mutating dhaK and acetobutylicum (Akawi et al., 2015; Srirangan et al., 2016a). coexpressing bktB and phaCAB from C. necator in the propanogenic E. Manipulating various genes involved in the glycerol dissimilation coli strain, resulting in a high specific P(3HB-co-3HV) yield of 66% of pathway potentially enhanced the production of propionate (Akawi dcw (Srirangan et al., 2016b). Moreover, inactivating respiratory gly- et al., 2015) and butanone (Srirangan et al., 2016a) from glycerol under cerol dissimilation via mutation of glpD in the strain coexpressing bktB various fermentation conditions. and phaCAB increased the 3-hydroxyvalerate (3-HV) content by 2.6- Small amounts (up to 154 mg/L) of 1-butanol were produced from fold to 18.5 mol%, which is the highest reported level for E. coli-based P glycerol by reconstructing the 1-butanol pathway from C. acet- (3HB-co-3HV) biosynthesis using an unrelated carbon source (Srirangan obutylicum in E. coli (Zhou et al., 2014). To use glycerol as a carbon et al., 2016b). Lastly, poly(3-hydroxyburyrate-co-3-hydroxyhexanoate) source, expression of native fdh1, encoding FDH, and inactivation of [P(3HB-co-3HH)] was synthesized in E. coli coexpressing phaAB from C. adhE, ldhA, and frdBC associated with side metabolite production aimed necator, and phaJ (encoding (R)-specific enoyl-CoA hydratase; PhaJ) to overcome the NADH deficit resulting from 1-butanol synthesis (Zhou and phaC from Aeromonas hydrophila, although P(3HB-co-3HH) pro- et al., 2014). Significantly higher 1-butanol titers of up to 6.9 g/L were duction was low on pure glycerol (Phithakrotchanakoon et al., 2013) obtained from crude glycerol in a ΔadhE ΔldhA ΔfrdA Δpta quadruple E. and was negligible when crude glycerol was used coli mutant expressing the 1-butanol pathway from C. acetobutylicum, (Phithakrotchanakoon et al., 2015). with phaA from C. necator and ter from T. denticola expressed in place of As is the case for 3-HP production, the regeneration of reducing thil and bcd-etfAB, respectively, by engineering parallel NADH re- equivalents and elimination of the B12-reliance for functional generation systems (Saini et al., 2017). Coexpression of the engineered

559 A.W. Westbrook et al. Biotechnology Advances 37 (2019) 538–568

⁎ native NADH-insensitive PDH complex (encoded by aceEF-lpdA ), and 5-HTP-producing strain. 5-HTP production was further enhanced by native pgl (encoding 6-phosphogluconolactonase; PGL) and zwf re- expressing engineered TPH2, i.e. TPH145 containing deletions of 145 spectively stimulated the decarboxylation of pyruvate to acetyl-CoA, N-terminal and 24 C-terminal amino acids, and by reducing the copy and increased flux through the PPP to regenerate NADPH, which was number of the plasmid containing L-tryptophan biosynthesis genes and converted to NADH via overexpressed native UDH (encoded by udhA), selecting different promoters to drive the synthesis and regeneration of resulting in a significant increase in the 1-butanol titer. Similarly, co- BH4. These strain engineering strategies resulted in the highest 5-HTP expression of native gldA and dhaKLM presumably increased NADH titer ever reported for bio-based production (5.1 g/L) (Wang et al., levels and, in turn, 1-butanol production, which could be further en- 2018). hanced by restricting carbon flux into the TCA cycle via promoter en- L-3,4-dihydroxyphenylalanine (L-DOPA), a precursor of dopamine gineering to reduce the expression of gltA to conserve acetyl-CoA (Saini used in the treatment of Parkinson’s disease and dopamine-responsive et al., 2017). dystonia, was synthesized from glycerol with an E. coli strain expressing E. coli was also engineered to produce 2,3-BDO isomers from gly- a modified Shikimate pathway (Das et al., 2018). L-DOPA is derived cerol. For the production of (R,R)-2,3-BDO, alsS, encoding α-acet- from L-tyrosine, which is the major effector of TyrR (L-tyrosine re- olactate synthase (AlsS) that converts pyruvate to (S)-2-acetolactate, pressor encoded by tyrR)-based repression of aromatic amino acid and alsD, encoding α-acetolactate decarboxylase (AlsD) that converts biosynthesis (Keseler et al., 2016), such that tyrR was mutated to in- (S)-2-acetolactate to (R)-acetoin, from B. subtilis were coexpressed with crease L-tyrosine levels. To further boost L-tyrosine overproduction, adh, encoding secondary alcohol dehydrogenase that converts (R)- feedback- resistant native 3-deoxy-D-arabino-heptulosonate synthase ⁎ ⁎ acetoin to (R,R)-2,3-BDO from C. beijerinckii, resulting in a (R,R)-2,3- (i.e., DAHP encoded by aroG ), shikimate kinase (i.e., AroL encoded by BDO titer of 9.5 g/L (Shen et al., 2012). Similarly, coexpression of budA, aroL), chorismate synthase (i.e., AroC encoded by aroC), 3-phos- encoding α-acetolactate decarboxylase (BudA) that converts (S)-2- phoshikimate 1-carboxyvinyltransferase (i.e., AroA encoded by aroA), acetolactate to (R)-acetoin, and budC, encoding 2,3-BDO dehy- and L-tyrosine aminotransferase (i.e., TyrB encoded by tyrB) were co- drogenase/acetoin reductase that converts (R)-acetoin to meso-2,3- expressed in a tyrR mutant also containing mutations to pykF (encoding BDO, from K. pneumoniae resulted in a meso-2,3-BDO titer of 6.9 g/L pyruvate kinase I; PykF) and serA (encoding phosphoglycerate dehy- from crude glycerol (Lee et al., 2012). drogenase; SerA) to block competing amino acid biosynthetic path- Production of up to 6.2 g/L 2-hydroxyisovalerate (an industrially ways. The direct conversion of L-tyrosine to L-DOPA was achieved by important surfactant, emulsifier, and intermediate for the synthesis of coexpressing native 4-hydroxyphenylacetate-3-hydrolase (i.e., HpaBC polymers, solvents, and fertilizers) from glycerol was achieved in E. coli encoded by hpaBC) in the L-tyrosine overproducing mutant, resulting in by exploiting the native L-valine biosynthetic pathway (Cheong et al., the production of up to 12.5 g/L L-DOPA, with a marked reduction in 2018). First, two pyruvate molecules were condensed to generate 2- acetate accumulation compared to previous studies in which L-DOPA acetolactate by AlsS from B. subtilis, followed by its sequential con- was derived from glucose (Das et al., 2018). version to 2,3-dihydroxyisovalerate, 2-ketoisovalerate, and finally 2- Itaconate is a versatile biological used in the production of resins, hydroxyisovalerate by native acetohydroxy-acid isomeroreductase (i.e., plastics, rubbers, paints, surfactants, and lubricants (Dwiarti et al., IlvC encoded by ilvC), native IlvD, and 2-hydroxyacid dehydrogenase 2007). Itaconate production from glycerol was demonstrated in E. coli (i.e., PanE endoded by panE) from L. lactis, respectively. Inactivation of expressing engineered cis-aconitate decarboxylase from Aspergillus ter- competing mixed-acid pathways resulted in a significant decrease in 2- reus (CadA encoded by cadA), which converts cis-aconitate (a TCA cycle hydroxyisovalerate production, highlighting the importance of these intermediate) to itaconate (Jeon et al., 2016). A library of synonymous pathways for maintaining the overall intracellular redox balance codon variants was constructed by introducing random point mutations (Cheong et al., 2018). in the first 10 codons of CadA, and inclusion body formation wasnearly β-carotene production from glycerol was achieved in E. coli by abolished when certain engineered CadA variants were expressed, reconstructing an alternative glycerol utilization pathway in a previously sulting in up to 4-fold increases in the itaconate titer, which reached 7.2 developed strain (Ye et al., 2016)(Guo et al., 2018). Glycerol was se- g/L in a fedbatch cultivation (Jeon et al., 2016). Moreover, inactivation quentially converted to D-glyceraldehyde and D-glycerate via aldehyde of icd and coexpression of codon optimized cadA from A. terreus, pyc reductase (i.e., Alrd encoded by alrd) and ALDH (i.e., AldH encoded by (encoding pyruvate carboxylase; Pyc) from C. glutamicum, and native aldH) from C. necator, respectively, and 2-phospho-D-glycerate was acnB (encoding aconitase; AcnB) and gltA resulted in a high itaconate derived from D-glycerate by a native glycerol kinase (i.e., GarK encoded titer of 43 g/L in a fedbatch cultivation (Chang et al., 2017). by garK). The synthetic glycerol utilization pathway simultaneously Deoxyviolacein, a drug with antagonistic activity against tumors, increased the rate of glycerol consumption and the supply of early Gram-positive bacteria, and fungal plant pathogens (Rodrigues et al., isoprenoid precursors, i.e., G3P and pyruvate, enabling the production 2014), was first produced from glucose in engineered E. coli expressing of 75 mg/L of β-carotene (Guo et al., 2018). a truncated deoxyviolacein gene cluster, i.e. vioABCE with vioD omitted, Production of 5-hydroxytryptophan (5-HTP), a precursor of ser- from C. violaceum (Rodrigues et al., 2013). The deoxyviolacein-produ- otonin used to treat depression, insomnia, and headaches, from glycerol cing strain was further modified via inactivation of araBAD, eliminating was recently demonstrated in E. coli (Wang et al., 2018). As a precursor L-arabinose metabolism, and, among various carbon sources, glycerol of 5-HTP biosynthesis, tetrahydrobiopterin (BH4) was produced in a was determined to be most effective for deoxyviolacein production up three-step pathway via GTP cyclohydrolase (GCHI), 6-pyruvate-tetra- to 1.6 g/L (Rodrigues et al., 2014). hydropterin synthase (PTPS), and sepiapterin reductase (SPR), encoded Valerenadiene is an anxiolytic compound derived from the medic- by mtrA from B. subtilis, human PTPS, and human SPR, respectively. 5- inal herb Valeriana officinalis, and has been produced in small quantities HTP was synthesized from BH4 and L-tryptophan by tryptophan hy- in engineered E. coli (Nybo et al., 2017). Coexpression of codon opti- droxylase I or II, i.e., TPH1/2 encoded by human TPH1/2, and BH4 was mized vds, encoding valerenadiene synthase from V. officinalis (VDS), regenerated via a two-step pathway comprised of pterin-4α-carbinola- and genes comprising the mevalonic acid pathway from S. cerevisiae mine dehydratase (PCD) and dihydropteridine reductase (DHPR), en- resulted in a valerenadiene titer of 62 mg/L from glycerol (Nybo et al., coded by human PCD and DHPR, respectively (Wang et al., 2018). To 2017). avoid the requirement for exogenous L-tryptophan supplementation, Small amounts (up to 38 mg/L) of toxic acrylic acid was synthesized the native L-tryptophan biosynthetic pathway, including engineered from glycerol using a previously constructed P(3HP)-producing mutant, ⁎ feedback resistant trpE (encoding tryptophan-insensitive anthranilate in which 3-HP-CoA was derived via coexpression of dhaB123 and gdrAB ⁎ ⁎ synthase subunit TrpE ) and aroH (encoding tryptophan-insensitive 3- from K. pneumoniae and pduP from S. typhimurium (Tong et al., 2016). ⁎ deoxy-7-phosphoheptulonate synthase AroH ), were coexpressed in the Acrylyl-CoA was derived from 3-HP-CoA via an enoyl-CoA hydratase

560 A.W. Westbrook et al. Biotechnology Advances 37 (2019) 538–568 from the propionyl-CoA synthase complex of Chloroflexus aurantiacus global metabolism may facilitate the derivation of superior production and the CoA moiety was subsequently removed by a putative en- hosts. dogenous CoA transferase (Tong et al., 2016). Serinol production (up to 3.3 g/L) from glycerol was achieved via 4.2. Strategies to enhance target metabolite production from glycerol expression of a bifunctional DHAP aminotransferase/dihy- drorhizobitoxine synthase from Bradyrhizobium elkanii, which mediates General strategies to enhance target metabolite production from DHAP conversion to serinol phosphate and subsequent depho- glycerol are universal among the hosts discussed herein, although the sphorylation (Andreeßen and Steinbüchel, 2012). specific approach and relative importance depend on the strain and product. For example, further improvements to 1,3-PDO production in 4. 4.0 Future perspectives E. coli or K. pneumoniae will likely hinge on improving the product tolerance of these strains and reducing inhibitory metabolite formation. 4.1. Selecting the appropriate production host for glycerol biorefinery In general, improving product tolerance may be achieved via cell membrane engineering (Tan et al., 2017; Zhao et al., 2003), directed Factors associated with the selection of a proper microbial platform evolution (Sandoval et al., 2015), or modulating the host stress re- for target metabolite production from glycerol include growth response via overexpression of heat shock proteins (Zingaro and quirements, product tolerance, pathogenicity, and genetic tractability. Papoutsakis, 2012). Reducing side metabolite formation in E. coli While K. pneumoniae is genetically amenable and has a high capacity for during 1,3-PDO production can be effectively achieved by inactivating the conversion of glycerol into diols and organic acids with minimal key genes (e.g. ptsH), whereas engineering of the TCA cycle in K. nutritional requirements, the pathogenicity limits its application to pneumoniae may be required to alleviate pyruvate accumulation in 2,3- biomanufacturing, particularly for human consumption products. On BDO-deficient mutants. On the other hand, engineering cofactor re- the other hand, E. coli possesses all of the same positive attributes, with generation systems that rely on co-substrate utilization should be a key even greater ease of genetic manipulation and a proven track record in approach to improve 1,3-PDO production in Citrobacter sp. and Lacto- biomanufacturing, and has been engineered to dissimilate glycerol into bacillus sp. In the case of Lactobacillus sp., partially activating dormant many non-native metabolites with similar efficiency to natural produ- fermentative or respiratory pathways for oxidative glycerol dissimila- cers. For example, using E. coli, 1,3-PDO is produced commercially from tion may also serve to restore redox poise. glucose (Celińska, 2010), and B12-independent production of 1,3-PDO A major disadvantage in using E. coli for the production of 3-HP and from glycerol has been achieved on a similar scale (Tang et al., 2009). P(3HP), or other oxidized metabolites derived from 3-HPA, is the re- Moreover, side metabolite formation appears less problematic in E. coli, quirement for exogenous B12. Biosynthesis of B12 in E. coli required the potentially due to its relatively stable TCA cycle, and the insignificant coexpression of 22 genes contained in six operons and three plasmids 2,3-BDO production during glycerol fermentation can simplify pur- (Ko et al., 2014), which, if implemented in a strain engineered to ification of 1,3-PDO. E. coli is also an ideal candidate for 3-HP pro- produce a second major metabolite (e.g. 3-HP), could pose a large duction due to its high production capacity and lack of redundant 1,3- burden on the host cell and compromise culture performance. As pre- + PDOORs, although the engineered strains often express B12-dependent viously outlined, NAD can be regenerated anaerobically via NADH:- GDHt which is a technical challenge difficult to overcome. quinone oxidoreductase II with supplemental nitrate, which may fa-

Certain Clostridium sp. are particularly well suited to the production cilitate 3-HP and P(3HP) production in strains expressing B12- of specific metabolites from glycerol, e.g. C. pasteurianum and C. bu- independent DhaB1 from C. butyricum. Alternatively, protein en- tyricum as 1-butanol and 1,3-PDO producers, respectively. However, gineering of DhaB1 to reduce or abolish its sensitivity to oxygen may they are obligate anaerobes that are recalcitrant to genetic manipula- circumvent the requirement of anaerobic operation, and this was suc- tion and, thus, may be restricted to the production of native products cessfully achieved with AdhE from E. coli for aerobic 1-propanol pro- with limited strain improvement. While Citrobacter sp. have not been duction (Choi et al., 2012). Yet, another possibility is the selection of a well characterized and, therefore, lack advanced genetic tools, certain production host that can naturally synthesize B12 such as Citrobacter sp., strains are attractive for glycerol biorefinery. In particular, C. werkmanii certain Lactobacillus sp., and K. pneumoniae. While K. pneumoniae is a readily utilizes co-substrates with a preference for glycerol, such that pathogen, attenuated strains lacking endogenous endotoxins have been blocking oxidative pathways for glycerol dissimilation to channel the developed through rational strain design (Huynh et al., 2015; Jung carbon flux into the reductive branch as well as engineering of cofactor et al., 2013), which is an approach that led to the development of non- regeneration systems that exploit glucose metabolism may be effective hemolytic Streptococcus equisimilis for commercial hyaluronic acid strategies in this host for target metabolite formation. Moreover, ex- production (Kim et al., 1996; Liu et al., 2011), and endotoxin-free E. coli ceptionally high 1,3-PDO yields from co-substrates have been reported for recombinant protein production (Mamat et al., 2015). Finally, the in cultures of C. werkmanii relative to common 1,3-PDO producer such application of mixed microbial cultures is another potential strategy to as Klebsiella sp. (Maervoet et al., 2012a, 2012b). Finally, Lactobacillus enhance the bioconversion of glycerol to value-added products, and has sp. are highly alcohol and acid tolerant and many have been granted been studied extensively for the production of 1,3-PDO (Jiang et al., GRAS status, making them suitable hosts for the production of biofuels 2018; Jiang et al., 2017; Sun et al., 2018; Zhou et al., 2017a, 2017b; and organic acids (Bosma et al., 2017). Moreover, the genomes of Zhou et al., 2018). Lactobacillus sp. are substantially smaller than other bacteria discussed While the strategies proposed herein may resolve certain limitations herein (Bosma et al., 2017). For example, K. pneumoniae MGH 78578 identified through various strain engineering approaches, a systems has a 5.32 Mb genome encoding 4,996 proteins, whereas L. reuteri DSM biology approach that incorporates metabolomics, proteomics, and 20016 has a 2 Mb genome encoding 1,917 proteins. Accordingly, it is transcriptomics may serve to elucidate the multiple layers of regulatory anticipated that minimizing side metabolite formation or overcoming architecture that orchestrate glycerol metabolism. Evidence suggests complex regulatory mechanisms that control flux distribution would be that regulatory mechanisms such as post-transcriptional regulation (i.e. significantly easier in L. reuteri. Note that certain species of this genus via non-coding RNAs), post-translational regulation (e.g. phosphoryla- are fastidious in their nutritional requirements, although other species tion), and allosteric regulation significantly affect metabolic flux dis- such as L. reuteri can proliferate in less complex media (Bosma et al., tribution, which is another important topic that has recently been re- 2017). Overall, while E. coli is the most preferred host for bio-based viewed (Liu et al., 2017). A systems level approach combined with next- production from glycerol, certain organisms are inherently specialized generation genome editing tools, such as the CRISPR-Cas9 system for the production of specific metabolites. Hence, the development of which has been implemented in various microorganisms (Choi and Lee, more sophisticated genetic tools and comprehensive understanding of 2016), is a logical step toward engineering microbial platforms for

561 A.W. Westbrook et al. Biotechnology Advances 37 (2019) 538–568 industrial-scale glycerol biorefinery. However, the functionality of the Biebl, H., 2001. Fermentation of glycerol by Clostridium pasteurianum — batch and CRISPR-Cas9 system in a given host might not necessarily result in continuous culture studies. Journal of Industrial Microbiology and Biotechnology 27 (1), 18–26. dramatic improvements to the frequency with which desired genome Blankschien, M.D., Clomburg, J.M., Gonzalez, R., 2010. Metabolic engineering of editing events occur. Moreover, it has been our experience that higher Escherichia coli for the production of succinate from glycerol. Metabolic Engineering order multiplexing of gene mutations may be limited by the efficiency 12 (5), 409–419. Bobik, T.A., Havemann, G.D., Busch, R.J., Williams, D.S., Aldrich, H.C., 1999. 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