Construction of a platform strain for anaerobic bioprocessing
and its application to biofuel production
Kenji OKANO
Department of Biotechnology, Graduate School of Engineering,Osaka University
ReportWe constructed a Lactobacillus plantarum double-knockout mutant for the L- andD-lactate dehydrogenase genes. By overexpressing pyruvate decarboxylase and alcohol
dehydrogenase in this double-knockout strain, we were able to produce 56.7 g/L of ethanolfrom 150 g/L of glucose (at 100 h of fermentation). Unlike conventional metabolicengineering hosts, such as Saccharomyces cerevisiae or Escherichia coli, ourdouble-knockout strain can be used to produce various value-added products, without
aeration or vigorous agitation. This anaerobic bioprocessing system should allow thedevelopment of economical bio-production processes.
Research aims
Metabolic engineering is a promising method for maximizing the production of specificmetabolites. S. cerevisiae and E. coli are common hosts for metabolic engineering. However,these microorganisms require an aerobic growth phase (with aeration and vigorous agitation)
before the fermentation production phase. As a consequence, bioproduction systems areoften economically unsustainable, particularly for the bulk production of chemicals orbiofuels. Moreover, because of the requirement for aeration during the growth phase, it isusually challenging to scale-up the fermentation process. This study aimed to construct aplatform microorganism for anaerobic bioprocessing (ABP), which requires neither aeration
nor vigorous agitation.
Methods
The bacterium L. plantarum maintains a similar growth rate under both aerobic andanaerobic conditions, which enables the growth-associated production of lactate without theneed for aeration and vigorous agitation. Therefore, L. plantarum is a suitable host for ABP.
However, the strain converts most of the fermented sugar into lactate.

To prevent the production of lactate, we disrupted both L- and D-lactate dehydrogenase
genes (ldhL1 and ldhD, respectively) in L. plantarum (Fig. 1). Moreover, to redirect thephosphoketolase pathway (which produces equimolar amounts of pyruvate and acetate from
pentose sugars) to the pentose phosphate pathway (which only produces pyruvate frompentose sugars), we replaced theGlucose

phosphoketolase gene (xpk1) with thetransketolase
gene(tkt)from(xylA, xylB)
Phosphoketolase

Lactococcus lactis. Next, to promote
ethanol fermentation, we introduced inthe ΔldhL1 ΔldhD strain pyruvatedecarboxylase (PDC) and alcohol
dehydrogenase (ADH) genes fromvarious origins. Finally, for the strain
showing the highest ethanol titer in
our initial tests, ethanol fermentation
was performed in a 1 L jar fermenterusing a glucose concentration of150 g/L.

XyloseArabinose

Xylulose-5-P(xpk1)
Acetyl-P

GAP

AcetateGlycolysis

R5P

Transketolase
(tkt)

GAP

S7P

GAP

E4P

F6PF6PFBP

GAP

PyruvateldhL
L-Lactic acidldhD

D-Lactic acid

DHAP(pdc, adh)
EtOH

Fig. 1. Metabolic pathways in the ABP platform
microorganism.

Results
We have previously reported the construction of a ΔldhD strain of L. plantarum, which
was confirmed to produce L-lactate with a high optical purity[1]. To construct the ΔldhL1ΔldhD strain, we disrupted the ldhL1 gene in the ΔldhD strain. As expected, the resulting
ΔldhL1 ΔldhD strain was unable to produce lactic acid. Remarkably, this strain could onlygrow under aerobic conditions, probably due to an inability to oxidize the NADH produced
through glycolysis. Furthermore, the disruption of the xpk1 gene in the ΔldhL1 ΔldhD strain
hindered cell growth on pentose. However, we were able to restore cell growth byintroducing the tkt gene into the disrupted xpk1 locus. Since L. plantarum can naturally use
arabinose but not xylose, we are now trying to introduce in the ΔldhL1 ΔldhD strain the xylA
and xylB genes to allow the strain to use xylose as an alternative carbon source.
To evaluate the ΔldhL1 ΔldhD strain as a platform microorganism for ABP, we expressedin this strain the PDC and ADH genes to promote ethanol production (Fig. 1). We tested thePDC genes from three different bacteria, Acetobacter pasteurianus (ApPDC), Zymomonas
mobilis (ZmPDC), and Lactobacillus florum (LfPDC), and the ADH genes from Oenococcus
oeni (OoADH) and Z. mobilis (ZmADH). We first performed ethanol fermentation using thedifferent PDC and ADH expression strains, and a glucose concentration of 100 g/L (Fig. 2).

Ethanol conc. (g/L)

35

1: OoADH32: ZmADH330

3: ApPDC
4: ApPDC + OoADH35: ApPDC + ZmADH32520

6: ZmPDC
7: ZmPDC + OoADH38: ZmPDC + ZmADH315105

9: LfPDC
10: LfPDC + OoADH311: LfPDC + ZmADH30123456789 10 11Fig. 2. Ethanol fermentation in ΔldhL1 ΔldhD strains expressing the indicated PDC andADH genes. All strains were grown for 48 h at 37°C in 100 mL flasks, using 30 mL ofmodified MRS medium supplemented with 100 g/L glucose and 15 g/L CaCO3.
While the strains expressing OoADH or ZmADH alone did not produce ethanol, thoseexpressing ApPDC or ZmPDC were able to produce ethanol. This result suggests that L.
plantarum endogenous ADH can catalyze the reduction of acetaldehyde to ethanol.
Moreover, the expression of ApPDC or ZmPDC restored the anaerobic growth of the ΔldhL1
ΔldhD strain. Our data also show that the co-expression of PDC and ADH further increased
the ethanol titer, with the ApPDC/OoADH strain producing the highest titer of ethanol ofconditions,
thestrain produced

56.7 g/L of ethanol in 100 h offermentation (Fig. 3). The observed
ethanol yield (g per g of consumedsugar) was 0.40, which was lower
than the theoretical yield of 0.51.
Since we found that lactate andacetate were still being produced,
further improvement of the metabolic
pathways in the ΔldhL1 ΔldhD strain
will be required to improve ethanol
yield and purity.

15060

12550

100407530502025100080 100 120

0204060
Time (h)Ethanol conc. (g/L)

UndertheseApPDC/OoADH

Glucose conc. (g/L)

33.0 g/L.
Encouraged by these results, we used the ApPDC/OoADH strain to perform a scaled-up
ethanol fermentation, using a 1 L jar fermenter and a glucose concentration of 150 g/L.

Fig. 3. Ethanol fermentation using the ApPDC/OoADH strain. Temperature and pH weremaintained at 37°C and 6.0, respectively.

Conclusion
In this study, we successfully produced ethanol using an engineered L. plantarum strain,
which was unable to produce lactate. Remarkably, the strain enabled the growth-associatedproduction of ethanol under anaerobic conditions and without aeration or vigorous agitation.
We believe that this ABP system using a platform microorganism can be further developed toallow the economical production of various chemicals and biofuels.
References
1) Okano, K., Uematsu, G., Hama, S., Tanaka, T., Noda, H., Kondo, A., and Honda, K. (2018)
Metabolic engineering of Lactobacillus plantarum for direct L-lactic acid production from raw
corn starch. Biotechnol. J. 13: 1700517.