Metabolic Engineering for Co-Production of Fuels and Chemicals Using a Single Microorganism

Authors: 
Park, S., Pusan National University
Seol, E., Pusan National University
Song, H., GS Caltex
Seung, D., GS Caltex
Sundara Sekar, B., Pusan National University
Ko, Y., Pusan National University

Microorganisms have evolved to optimize their growth, not for the formation of metabolite(s) useful to human. Microbial growth occurs with or through generation of multiple metabolites, but metabolic engineering usually focuses on the production of a ‘single’ compound by re-designing and re-directing metabolic pathways [1]. This often seriously interferes with cellular metabolisms, hurts cell growth and viability, and makes the microorganism to function inefficiently as microbial cell factory. Production of properly-selected, multiple metabolites, instead of a single metabolite, can be a useful strategy to improve the performance of microbial cell factory by alleviating cellular stresses caused by metabolic engineering. Co-production can offer flexible balancing of the in vivo metabolic flux and easier maintenance of cellular homeostasis including redox state. To demonstrate the potential of the co-production strategy, we have studied Klebsiella pneumoniae for co-production of 3-hydroxypropionic acid (3-HP) and 1,3-propanediol (1,3-PDO) or 1,3-PDO and 2,3-butanediol (2,3-BDO) from glycerol, and Escherichia coli for co-production of H2 and ethanol or 3-HP from glucose. Co-production of 3-HP and 1,3-PDO made the generation and consumption of NAD(H) well-balanced, reduced the accumulation of the toxic intermediate, 3-hydroxypropionaldehyde, and resulted in high glycerol-to-products yield (>0.8) at high titer (>70 g/L) [2]. Co-production of 1,3-PDO and 2,3-BDO by K. pneumoniae alleviated metabolic traffic at the pyruvate node, reduced acetate accumulation, and enabled the maintenance of redox balance. With glycerol as sole carbon source or along with glucose, >100 g/L products (1,3-PDO plus 2,3-BDO) could be obtained. For co-production of H2 and ethanol from glucose, carbon flux was diverted to the pentose phosphate (PP) pathway from the Embden-Meyerhof-Parnas (EMP) pathway by over-expressing the two major enzymes of PP pathway, Zwf and Gnd, and by disrupting or down-regulating Pfk or Pgi of EMP pathway [3]. Increase in NAD(P)H supply enhanced ethanol production, and H2 and ethanol could be obtained with the theoretical maximum yields and negligible acetate. During the development of various biocatalysts, to meet the specific requirement for each biocatalyst and/or carbon substrate used, deletion and amplification of many pathways were made by employing carefully-selected enzymes and gene expression systems (dynamic promoters, 5’UTR engineering, etc). This lecture also discusses the challenges of the co-production strategy.

 References

  1.  Kumar, Vinod, Somasundar Ashok, and Sunghoon Park. “Recent advances in biological production of 3-hydroxypropionic acid.” Biotechnology advances 31 (2013): 945-961.
  2. Kumar, Vinod, et al. “Simultaneous production of 3-hydroxypropionic acid and 1, 3-propanediol from glycerol using resting cells of the lactate dehydrogenase-deficient recombinant Klebsiella pneumoniae overexpressing an aldehyde dehydrogenase.” Bioresource technology 135 (2013): 555-563.
  3. Seol, E., Sekar, B. S., Raj, S. M. and Park, S. “Co-production of hydrogen and ethanol from glucose by modification of glycolytic pathways in Escherichia coli – from Embden-Meyerhof-Parnas pathway to pentose phosphate pathway.” Biotechnology Journal 11 (2016): 249–256

Acknowledgement

This work was supported by the Advanced Biomass R&D Center (ABC) of Global Frontier Project funded by the Ministry of Science, ICT and Future Planning (ABC-2011-0031361). The authors are grateful also to the BK21 Plus program at Pusan National University.