Isopropanol production via the thermophilic bioconversion of sugars and syngas using metabolically engineered Moorella thermoacetica

Abstract

Background: Isopropanol (IPA) is a commodity chemical used as a solvent or raw material for polymeric products, such as plastics. Currently, IPA production depends largely on high-CO₂-emission petrochemical methods that are not sustainable. Therefore, alternative low-CO₂ emission methods are required. IPA bioproduction using biomass or waste gas is a promising method.

Results: Moorella thermoacetica, a thermophilic acetogenic microorganism, was genetically engineered to produce IPA. A metabolic pathway related to acetone reduction was selected, and acetone conversion to IPA was achieved via the heterologous expression of secondary alcohol dehydrogenase (sadh) in the thermophilic bacterium. sadh-expressing strains were combined with acetone-producing strains, to obtain an IPA-producing strain. The strain produced IPA as a major product using hexose and pentose sugars as substrates (81% mol-IPA/mol-sugar). Furthermore, IPA was produced from CO, whereas acetate was an abundant byproduct. Fermentation using syngas containing both CO and H₂ resulted in higher IPA production at the specific rate of 0.03 h^-1. The supply of reducing power for acetone conversion from the gaseous substrates was examined by supplementing acetone to the culture, and the continuous and rapid conversion of acetone to IPA showed a sufficient supply of NADPH for Sadh.

Conclusions: The successful engineering of M. thermoacetica resulted in high IPA production from sugars. M. thermoacetica metabolism showed a high capacity for acetone conversion to IPA in the gaseous substrates, indicating acetone production as the bottleneck in IPA production for further improving the strain. This study provides a platform for IPA production via the metabolic engineering of thermophilic acetogens.

Keywords: Biomass; Isopropanol production; Secondary alcohol dehydrogenase; Syngas; Thermophilic acetogen.

Fig. 1

Designed IPA production pathway and NADPH supply. A Pathway for the synthesis of IPA from sugars and gaseous substrates. A key intermediate, acetyl-CoA, was processed by heterologously expressed enzymes for acetone synthesis (blue color) and Sadh (red color). Acetate, provided by the native pathway, was used to receive CoA from acetoacetyl-CoA. One of the genes, pduL2, in the acetate pathway was disrupted to enhance the carbon flow to IPA. One molecule of NADPH is required to convert acetone into IPA. Thl, thiolase; CtfAB, CoA transferase; Adc, acetoacetate decarboxylase; PduL1 and PduL2, phosphotransacetylase; Ack, acetate kinase. B NADPH supply using reducing equivalents derived from sugar metabolism. The sugar metabolism provided reduced form of ferredoxin (Fd²⁻) and NADH. The NfnAB complex transferred electrons from Fd²⁻ and NADH to NADP⁺, simultaneously. C In the presence of H₂, NADPH was formed via two pathways. One was via Fd²⁻ and NADH. The HydABC complex transferred electrons from H₂ to Fd and NAD⁺, followed by the transfer by NfnAB complex. The other pathway was a direct electron transfer from H₂ to NADP⁺

Fig. 2

Introduction of sadh in M. thermoacetica. A Schematic representation of plasmid construction and sadh introduction in place of pduL2. B Agarose gel electrophoresis following PCR amplification of the pduL2 region of the host and a recombinant strain. M, DNA size marker; 1, the pduL2::sadh strain; 2, the ∆pyrF strain. The size of the amplified region was shifted to 2.6 kb in the pduL2::sadh strain, consistent with the DNA construct, which was originally 0.9 kb in the ∆pyrF strain. C A plausible metabolic pathway showing cofactor supply for ethanol production from hexose. Reducing equivalents NADH and reduced Fd were converted into NADPH via enzymatic electron confurcation. NADPH is a cofactor for Sadh. D Culture profile of the pduL2::sadh strain supplemented with fructose as the substrate. E Acetone supplementation to the culture of the pduL2::sadh strain. The condition was the same as in D. Data are presented as the mean with SDs of two biological replicates in D, E. Most error bars are smaller than the symbols of data plots

Fig. 3

Introduction of genes for IPA production in M. thermoacetica. A Schematic representation of the synthetic IPA operon encoding enzymes for IPA production from acetyl-CoA. The reactions by enzymes derived from corresponding genes are shown in Fig. 1. B Schematic representation of the plasmid construction and introduction of the synthetic IPA operon in place of pduL2. C Agarose gel electrophoresis, following PCR amplification of the pduL2 region of the host and recombinant strains. M, DNA size marker; 1, the ∆pyrF strain; 2, the plasmid pHM71 (positive control); 3–8, candidates of the pduL2::IPA strain. The size of the amplified region was shifted to 6.5 kb in the pduL2::IPA strain, consistent with the DNA construct, which was originally 0.9 kb in the ∆pyrF strain. A candidate clone (lane No. 3) showed a faint band with the same migration as the ∆pyrF strain, which indicated a mixed population. This clone was excluded. The other clones showed the same culture profile in the following fermentation analysis

Fig. 4

Culture profiles of the pduL2::IPA strain when supplemented with hexose (fructose) and pentose (xylose) sugars. The consumption of fructose (A) or xylose (B) was monitored and products including acetate, acetone, formate, and IPA, in the culture supernatant were measured at each time point. Dry cell weight was calculated according to the OD. Data are presented as the mean with SDs of three biological replicates. Most error bars are smaller than the symbols of data plots

Fig. 5

Culture profiles of the pduL2::IPA strain when supplemented with gaseous substrates, CO (A) or syngas (CO:H₂ = 1:1) (B). Products including acetate, acetone, formate, and IPA, in the culture supernatant were measured at each time point. Dry cell weight was calculated according to the OD. Data are presented as the mean with SDs of three biological replicates

Fig. 6

Culture profiles of the pduL2::IPA strain when supplemented with syngas for testing acetone reduction. Acetone (50 mM) was added to the culture medium in the culture vial filled with syngas (CO:H₂ = 1:2), followed by inoculation and starting culture. Products including acetate, acetone, formate, and IPA, in the culture supernatant were measured at each time point. Dry cell weight was calculated according to the OD. Data are presented as the mean with SDs of three biological replicates. Some error bars are smaller than the symbols of data plots