Metabolic Engineering of a Glycerol-Oxidative Pathway in Lactobacillus panis PM1 for Utilization of Bioethanol Thin Stillage: Potential To Produce Platform Chemicals from Glycerol

Abstract

Lactobacillus panis PM1 has the ability to produce 1,3-propanediol (1,3-PDO) from thin stillage (TS), which is the major waste material after bioethanol production, and is therefore of significance. However, the fact that L. panis PM1 cannot use glycerol as a sole carbon source presents a considerable problem in terms of utilization of this strain in a wide range of industrial applications. Accordingly, L. panis PM1 was genetically engineered to directly utilize TS as a fermentable substrate for the production of valuable platform chemicals without the need for exogenous nutrient supplementation (e.g., sugars and nitrogen sources). An artificial glycerol-oxidative pathway, comprised of glycerol facilitator, glycerol kinase, glycerol 3-phosphate dehydrogenase, triosephosphate isomerase, and NADPH-dependent aldehyde reductase genes of Escherichia coli, was introduced into L. panis PM1 in order to directly utilize glycerol for the production of energy for growth and value-added chemicals. A pH 6.5 culture converted glycerol to mainly lactic acid (85.43 mM), whereas a significant amount of 1,3-propanediol (59.96 mM) was formed at pH 7.5. Regardless of the pH, ethanol (82.16 to 83.22 mM) was produced from TS fermentations, confirming that the artificial pathway metabolized glycerol for energy production and converted it into lactic acid or 1,3-PDO and ethanol in a pH-dependent manner. This study demonstrates the cost-effective conversion of TS to value-added chemicals by the engineered PM1 strain cultured under industrial conditions. Thus, application of this strain or these research findings can contribute to reduced costs of bioethanol production.T hin stillage (TS) is a major fermentation residue from the dry-grind process of bioethanol production. Its downstream treatment, including making a condensed form for animal feed, is an energy-and cost-intensive process (1), while its utilization as animal feed is hardly a high-value application of this industrial by-product and hence does not significantly reduce the cost of ethanol production. TS contains a variety of complex nutrients, including various carbohydrates, minerals, and amino acids. Glycerol (up to 2%) is generated in TS during bioethanol production regardless of feedstock (i.e., sugar cane or corn) (2). Glycerol is at a higher reduced state than carbohydrates (i.e., fermentable sugars). The reduced nature of glycerol allows synthesis of fuels and other reduced chemicals at higher yields with minimal by-products compared to common sugars (e.g., glucose and xylose); however, the high degree of reduction in glycerol makes it difficult for microorganisms to utilize under anaerobic conditions. A few bacteria, including Klebsiella, Enterobacter, and Clostridium, have been exploited to bioconvert glycerol to industrially relevant chemicals (e.g., 1,3-propanediol [1,3-PDO], 3-hydroxypropionic acid, butanol, and ethanol) (3-7). Since these organisms are classified as opportunistic pathogens, industrial applications of these strains may pose issues, particularly re...

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