2,3-Butanediol (2,3-BD) is a platform chemical with vast industrial applications; particularly for its use in the production of 1,3-butadiene (1,3-BD), the monomer from which synthetic rubber is manufactured. Fermentative production of 2,3-BD is hampered by cost of food-based substrates; low 2,3-BD titer, yield and productivity stemming from formation of competing products such as exopolysaccharides (EPS), ethanol, lactic, formic and acetic acids. The objectives of this study were conceived to examine use of process design, alternative substrates and metabolic engineering to enhance 2,3-BD production.
The first objective examined the impact of yeast extract, tryptone, ammonium acetate, ammonium sulfate, glycerol, inoculum size and fermentation temperature on 2,3-BD production by Paenibacillus polymyxa. Tryptone, temperature and inoculum size significantly influenced 2,3-BD production; and optimization of these factors increased 2,3-BD production by P. polymyxa from ~27 g/L to 51.1 g/L and 47 g/L to 68.5 g/L in batch and fed-batch cultures, respectively. The second objective focused on understanding 2,3-BD-mediated feedback inhibition during 2,3-BD fermentation. The response of P. polymyxa to high 2,3-BD concentrations during growth and 2,3-BD fermentation was evaluated due to inability of P. polymyxa to produce >6% 2,3-BD in fed-batch cultures. Cultures challenged with levo-2,3-BD (20, 40 and 60 g/L) at 0 h of fermentation inhibited the growth of P. polymyxa in a concentration dependent manner. Furthermore, when P. polymyxa was challenged with incremental 2,3-BD concentrations (20, 40 and 60 g/L at 12, 24 and 36 h, respectively), 2,3-BD was reconverted to acetoin (the precursor from which 2,3-BD is biosynthesized) when 2,3-BD concentration reached 60 g/L, possibly to alleviate 2,3-BD toxicity.
The third objective investigated the ability of P. polymyxa to use LB-based agricultural residue, wheat straw hydrolysate (WSH), for 2,3-BD production. Prior to testing the fermentability of WSH, the ability of P. polymyxa to co-metabolize representative LB mixed sugars (glucose, xylose and arabinose) was evaluated. The results showed that P. polymyxa simultaneously co-metabolized the mixed sugar components of LB to 2,3-BD. Batch fermentations using 60%, 80%, and 100% WSH, showed that 2,3-BD production was 32, 31 and 23 g/L, respectively, and were comparable to 32 g/L obtained in the glucose-based control.
The last objective explored a metabolic engineering strategy to deactivate the EPS production pathway of P. polymyxa and drastically reduce EPS production. The study identified levansucrase gene that encodes levansucrase, the enzyme responsible for EPS biosynthesis in P. polymyxa. The levansucrase gene was successfully disrupted, and the resulting P. polymyxa levansucrase null mutant showed 34% and 54% increases in growth in sucrose and glucose media, respectively. Additionally, the P. polymyxa levansucrase null mutant grown in sucrose and glucose media produced 6.4- and 2.4-fold lower EPS, respectively, than that produced by the wildtype. The observed decrease in EPS formation by the levansucrase null mutant may be a direct cause of the 4-27% increase in 2,3-BD yield, and 4-128% increase in 2,3-BD productivity observed during 2,3-BD fermentation. Collectively, our results show that P. polymyxa levansucrase null mutant has potential for improving the economics of large-scale microbial 2,3-BD production.