Metabolic engineering of Zymomonas mobilis for co-production of D-lactic acid and ethanol using waste feedstocks of molasses and corncob residue hydrolysate

Abstract

Lactate is the precursor for polylactide. In this study, a lactate producer of Z. mobilis was constructed by replacing ZMO0038 with LmldhA gene driven by a strong promoter PadhB, replacing ZMO1650 with native pdc gene driven by Ptet, and replacing native pdc with another copy of LmldhA driven by PadhB to divert carbon from ethanol to D-lactate. The resultant strain ZML-pdc-ldh produced 13.8 ± 0.2 g/L lactate and 16.9 ± 0.3 g/L ethanol using 48 g/L glucose. Lactate production of ZML-pdc-ldh was further investigated after fermentation optimization in pH-controlled fermenters. ZML-pdc-ldh produced 24.2 ± 0.6 g/L lactate and 12.9 ± 0.8 g/L ethanol as well as 36.2 ± 1.0 g/L lactate and 40.3 ± 0.3 g/L ethanol, resulting in total carbon conversion rate of 98.3% ± 2.5% and 96.2% ± 0.1% with final product productivity of 1.9 ± 0.0 g/L/h and 2.2 ± 0.0 g/L/h in RMG5 and RMG12, respectively. Moreover, ZML-pdc-ldh produced 32.9 ± 0.1 g/L D-lactate and 27.7 ± 0.2 g/L ethanol as well as 42.8 ± 0.0 g/L D-lactate and 53.1 ± 0.7 g/L ethanol with 97.1% ± 0.0% and 99.1% ± 0.8% carbon conversion rate using 20% molasses or corncob residue hydrolysate, respectively. Our study thus demonstrated that it is effective for lactate production by fermentation condition optimization and metabolic engineering to strengthen heterologous ldh expression while reducing the native ethanol production pathway. The capability of recombinant lactate-producer of Z. mobilis for efficient waste feedstock conversion makes it a promising biorefinery platform for carbon-neutral biochemical production.

A new Zymomonas mobilis platform strain for the efficient production of chemicals

“…5). Data are derived from or partial knockout however already shows beneficial effects on the yields of products other than ethanol [8,[14][15][16][17]. The most promising approach was presented by Liu et al [15] who expressed pdc of Z. mobilis from a plasmid and subsequently deleted the original pdc locus (ZMO1360) from the chromosome.…”

“…However, to our knowledge, this approach has not been reported so far in peer review publications and it does not easily allow a switch to another product pathway as is possible with our platform strain sGB027. A knockdown or partial knockout however already shows beneficial effects on the yields of products other than ethanol [ 8 , 14 – 17 ]. The most promising approach was presented by Liu et al [ 15 ] who expressed pdc of Z. mobilis from a plasmid and subsequently deleted the original pdc locus (ZMO1360) from the chromosome.…”

“…While the wild type only has a narrow substrate spectrum, being able to metabolize glucose, fructose and sucrose, it has many desirable for high ethanol yield and productivity but hampers the production of other chemicals of interest. There is an increasing spectrum of products synthesized by genetically modified Z. mobilis strains, including malate, lactate, alanine, acetoin or the heterologous products 2,3-butanediol and isobutanol, but ethanol remains an essential by-product of all those strains, thereby reducing the yields for the respective product to varying degrees [8][9][10][11][12][13][14][15].…”

“…To reduce the amount of ethanol as a by-product, the activity of the enzyme pyruvate decarboxylase (PDC; GenBank: AAV89984.1), which converts pyruvate to acetaldehyde, has to be eliminated or strongly reduced. However, a complete knock-out of pdc (ZMO1360) in the wild type (WT) background proved impossible leading to different strategies to reduce its activity [8,[14][15][16][17]. Most studies were only able to generate heterogeneous knockout strains, which retained significant PDC activity.…”

“…To account for the redox imbalance caused by the lack of ethanol production, other pathways converting pyruvate towards alternative products (2,3-butanediol, isobutanol, lactate) were introduced by overexpressing the corresponding enzymes. For example, expression of the lactate dehydrogenase from E. coli , resulted in lactate production of about 70% of the theoretical maximum [ 15 ] which is significantly higher than lactate production achieved in the wild type background with lactate dehydrogenase expressed either from a plasmid or integrated into the chromosome (20% and 15% of the theoretical maximum, respectively) [ 9 , 14 ]. A complete removal of the pdc gene was described in a patent US 20190153483A1 [ 18 ].…”