Metabolically engineered Caldicellulosiruptor bescii as a platform for producing acetone and hydrogen from lignocellulose

Abstract

The production of volatile industrial chemicals utilizing metabolically engineered extreme thermophiles offers the potential for processes with simultaneous fermentation and product separation. An excellent target chemical for such a process is acetone (T b = 56°C), ideally produced from lignocellulosic biomass. Caldicellulosiruptor bescii (T opt 78°C), an extremely thermophilic fermentative bacterium naturally capable of deconstructing and fermenting lignocellulose, was metabolically engineered to produce acetone. When the acetone pathway construct was integrated into a parent strain containing the bifunctional alcohol dehydrogenase from Clostridium thermocellum, acetone was produced at 9.1 mM (0.53 g/L), in addition to minimal ethanol 3.3 mM (0.15 g/L), along with net acetate consumption. This demonstrates that C. bescii can be engineered with balanced pathways in which renewable carbohydrate sources are converted to useful metabolites, primarily acetone and H 2 , without net production of its native fermentation products, acetate and lactate.

Structural and Functional Analysis of a Multimodular Hyperthermostable Xylanase-Glucuronoyl Esterase from Caldicellulosiruptor kristjansonii

“…There might also be a possibility that the cadherin domain has carbohydrate binding properties that our focus on CAZy-annotated domains has neglected. The development of genetic engineering tools for the related C. bescii may also provide an alternative expression system to yield full-length Ck Xyn10C-GE15A, which would enable investigation of potential intramolecular synergy …”

Thermophilic microbial deconstruction and conversion of natural and transgenic lignocellulose

“…In comparison with T. saccharolyticum a nd C. thermocellum , C. bescii has the advantage of being able to degrade and metabolize both the six and five‐carbon sugars contained in cellulose and hemicellulose, but C. bescii does not possess the native ethanol production capability of the other two microbes. Metabolic engineering for production of commodity chemicals from C. bescii has largely targeted volatile chemicals, with success in producing ethanol (up to 3.5 g/l) and acetone (up to 0.5 g/l) (Williams‐Rhaesa et al ., 2018a; Straub et al ., 2020b). Metabolic engineering efforts have not yet achieved comparable titers to those reported for C. thermocellum and T. saccharolyticum ; with the most success involving the elimination of lactate production and expression of non‐native enzymes leading to titers of industrially relevant products so far <5 g/l (Lipscomb et al ., 2016; Williams‐Rhaesa et al ., 2018a; Straub et al ., 2020b).…”

“…Metabolic engineering for production of commodity chemicals from C. bescii has largely targeted volatile chemicals, with success in producing ethanol (up to 3.5 g/l) and acetone (up to 0.5 g/l) (Williams‐Rhaesa et al ., 2018a; Straub et al ., 2020b). Metabolic engineering efforts have not yet achieved comparable titers to those reported for C. thermocellum and T. saccharolyticum ; with the most success involving the elimination of lactate production and expression of non‐native enzymes leading to titers of industrially relevant products so far <5 g/l (Lipscomb et al ., 2016; Williams‐Rhaesa et al ., 2018a; Straub et al ., 2020b). This is in part due to the lack of native ethanol production pathways that could be optimized; non‐native product titers in C. bescii (ethanol and acetone) are comparable to C. thermocellum (such as n‐butanol) (Tian et al ., 2019).…”

Genome-Scale Metabolic Model of Caldicellulosiruptor bescii Reveals Optimal Metabolic Engineering Strategies for Bio-based Chemical Production

“…Engineered C. bescii strains are capable of producing ethanol ( 7 – 11 ) or acetone and H 2 ( 12 ) by fermenting simple sugars (i.e., cellobiose and maltose), crystalline cellulose, or plant biomass (i.e., switchgrass and poplar) as the sole carbon and energy source. Wild-type C. bescii does not contain genes required for ethanol production, so foreign genes have been introduced through a uracil auxotrophy system enabled by development of pyrFA or pyrE mutants for the construction of engineered strains ( 13 , 14 ).…”