Biotechnology Advances 37 (2019) 538–568 Contents lists available at ScienceDirect Biotechnology Advances journal homepage: www.elsevier.com/locate/biotechadv 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 bot- tlenecks 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-bu- tanediol; 3-HH, 3-hydroxyhexanoate; 3-HP, 3-hydroxypropionic acid; 3-HV, 3-hydroxyvalerate; (R)-3-HV-CoA, (R)-3-hydroxyvaleryl-CoA; ALDH, aldehyde dehy- drogenase; ACE, Allele-Coupled Exchange; AOR, aldehyde oxidoreductase; asRNA, antisense RNA; ATP, adenosine Triphosphate; cAMP, cyclic adenosine mono- phosphate; 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, poly- hydroxyalkanoate; 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 decarbox- ylase; 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 chemi- cals 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