Engineering Yarrowia lipolytica for the production of β-carotene by carbon and redox rebalancing

Abstract

Background: β-Carotene is a natural product that has garnered significant commercial interest. Considerable efforts have been made to meet such demand through the metabolic engineering of microorganisms, yet there is still potential for improvement. In this study, engineering approaches including carbon and redox rebalancing were used to maximize β-carotene production in Yarrowia lipolytica.

Results: The initial production level was increased by iterative overexpression of pathway genes with lycopene inhibition removal. For further improvement, two approaches that redirect the central carbon pathway were evaluated to increase NADPH regeneration and reduce ATP expenditure. Pushing flux through the pentose phosphate pathway and introducing NADP⁺-dependent glyceraldehyde-3-phosphate dehydrogenase were found to be more effective than the phosphoketolase-phosphotransacetylase (PK-PTA) pathway. Furthermore, flux to the lipid biosynthesis pathway was moderately increased to better accommodate the increased β-carotene pool, resulting in the production level of 809.2 mg/L.

Conclusions: The Y. lipolytica-based β-carotene production chassis was successfully developed through iterative overexpression of multiple pathways, central carbon pathway engineering and lipid pathway flux adjustment. The approach presented here provides insights into future endeavors to improve microbial terpenoid production capability.

Keywords: Yarrowia lipolytica; Central carbon pathway engineering; β-carotene.

Fig. 1

Construction of the base strain for β-carotene production. A Scheme illustrating the engineered metabolic nodes of β-carotene production, including synthetic and mevalonate pathways. Dashed arrows represent multiple metabolic reactions in between. CarRP, phytoene synthase/lycopene cyclase; CarB, phytoene dehydrogenase; ERG20, farnesyl pyrophosphate synthetase; GGPPS, geranyl geranyl diphosphate synthase; HMGR, hydroxymethylglutaryl-CoA reductase; ERG12, mevalonate kinase. B Carotenoid production and specific growth rate of derivative strains. Each plus sign represents a single round of integration of corresponding gene expression cassettes into the genome. Error bars represent the standard deviations of three biological replicates

Fig. 2

Elimination of lycopene inhibition and recovery of production level. A The CRISPR-cas9 system by which Y27R mutation was introduced into the YB4 strain. B Normalized fluorescence intensity of carRP-hrGFP fusion protein expressing strains. The star, with its distinct colors, indicates different codons for the Y27R mutation. The double tilde signs represent the discretization of the x-axis for clear comparison between weak fluorescent strains. C Carotenoid production and specific growth rate of derivative strains. Each plus sign represents a single round of integration of corresponding gene expression cassettes into the genome or deletion of complete CDS from the genome. Error bars represent the standard deviations of three biological replicates. The red dashed line indicates the β-carotene titer (mg/L) of YB4 from Fig. 1B

Fig. 3

Central carbon redistribution to regenerate NADPH and reduce ATP expenditure. A Scheme illustrating central carbon metabolic engineering strategies. ZWF1, glucose-6-phosphate dehydrogenase; GND1, 6-phosphogluconate dehydrogenase; TKL1, transketolase; TAL1, transaldolase; PFK, phosphofructokinase; GPD, glycerolglyceraldehyde-3-phosphate dehydrogenase; GapC, glyceraldehyde-3-phosphate dehydrogenase from Clostridium acetobutylicum; GapN, glyceraldehyde-3-phosphate dehydrogenase from C. acetobutylicum; PK, phosphoketolase from C. acetobutylicum; PTA, phosphotransacetylase from Thermoanaerobacterium saccharolyticum; IPP, isopentenyl diphosphate. B Carotenoid production of PK-PTA pathway strains. The brown dashed line indicates the β-carotene titer (mg/L) of YB14 from Fig. 3B. C Carotenoid production and specific growth rate of PP pathway and glycolysis engineered strains. Each plus sign represents a single round of integration of corresponding gene expression cassettes into the genome or deletion of complete CDS from the genome. The green dashed line indicates the β-carotene titer (mg/L) of YB14 from Fig. 3C. Error bars indicate standard deviations of biological triplicates

Fig. 4

Flux adjustment of lipid biosynthesis pathway. A Scheme illustrating flux divergence of lipid biosynthesis and carotenoid biosynthesis pathway from acetyl-CoA (B) Carotenoid production and specific growth rate of lipid biosynthesis overexpression strains. Each plus sign represents a single round of integration of corresponding gene expression cassettes or replacement of promoter into the genome. The blue dashed line indicates the β-carotene titer (mg/L) of YB19 from Fig. 3C. Error bars represent the standard deviations of three biological replicates