Efficient production of lycopene in Saccharomyces cerevisiae by enzyme engineering and increasing membrane flexibility and NAPDH production

Hong, Juhyun; Park, Seong-Hee; Kim, Sujin; Kim, Seon-Won; Hahn, Jinyong

doi:10.1007/s00253-018-9449-8

Cited by 99 publications

(79 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The proteins related to lipid metabolism (such as Acc1p, Ino1p and Cyb5p) and ergosterol biosynthetic process (Erg1p, Erg4p, Erg6p, Erg20p, Erg24p, and Erg25p) were simultaneously enriched in YBX-01 strain (Additional file 1: Table S1). This confirmed the occurrence of membrane stress in recombinant yeast and the necessity of improving membrane flexibility for efficient synthesizing carotenoid [22,31]. We also observed the amounts of cell wall synthesis proteins (Mpg1p, Crh1p, and Pst1p) raised in YBX-01.…”

Section: Mining Potential Transporters For β-Carotene Effluxsupporting

confidence: 76%

“…In the presence of acetate supplementation, OLE1 overexpression resulted in the slight improvement of biomass (from 21.2 to 22.5 [OD 600 ]) and intracellular β-carotene production (from 132.5 to 149.8 mg/L) simultaneously (Fig. 6 b), probably due to the alleviation of membrane stress and decrement of product toxicity on the host strain [ 22 ]. More importantly, 18.8% increased secretion of β-carotene (10.1 mg/L) was observed in the YBX-20 strain compared with the strain YBX-SNQ2 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Overproduction of efflux pumps or heterogenous biofuels anchor in the cell membrane can disorganize its structural integrity that impairs vital functions [16]. There are several works demonstrating that improving membrane fluidity by enriching intracellular unsaturated fatty acids (UFAs) can decrease the membrane stress [22,31,44]. Considering that membrane function greatly affects the secretion efficiency of products, we hypothesized that improving the membrane fluidity of host cells may be beneficial to β-carotene secretion.…”

Section: Improving Membrane Fluidity To Enhance β-Carotene Secretionmentioning

confidence: 99%

“…Thirdly, the overproduction of membrane proteins embedded in the cell membrane would increase the rigidity of the cell membrane and disturb its normal function, one consequence is decreasing cell growth and the transport capacity [21]. Maintaining the normal function of the cell membrane is therefore necessary to efficient export products, such as improving membrane fluidity [22].…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Engineering endogenous ABC transporter with improving ATP supply and membrane flexibility enhances the secretion of β-carotene in Saccharomyces cerevisiae

Lin

Cheng

et al. 2020

Biotechnol Biofuels

View full text Add to dashboard Cite

Background Product toxicity is one of the bottlenecks for microbial production of biofuels, and transporter-mediated biofuel secretion offers a promising strategy to solve this problem. As a robust microbial host for industrial-scale production of biofuels, Saccharomyces cerevisiae contains a powerful transport system to export a wide range of toxic compounds to sustain survival. The aim of this study is to improve the secretion and production of the hydrophobic product (β-carotene) by harnessing endogenous ABC transporters combined with physiological engineering in S. cerevisiae. Results Substrate inducibility is a prominent characteristic of most endogenous transporters. Through comparative proteomic analysis and transcriptional confirmation, we identified five potential ABC transporters (Pdr5p, Pdr10p, Snq2p, Yor1p, and Yol075cp) for β-carotene efflux. The accumulation of β-carotene also affects cell physiology in various aspects, including energy metabolism, mitochondrial translation, lipid metabolism, ergosterol biosynthetic process, and cell wall synthesis. Here, we adopted an inducible GAL promoter to overexpress candidate transporters and enhanced the secretion and intracellular production of β-carotene, in which Snq2p showed the best performance (a 4.04-fold and a 1.33-fold increase compared with its parental strain YBX-01, respectively). To further promote efflux capacity, two strategies of increasing ATP supply and improving membrane fluidity were following adopted. A 5.80-fold increase of β-carotene secretion and a 1.71-fold increase of the intracellular β-carotene production were consequently achieved in the engineered strain YBX-20 compared with the parental strain YBX-01. Conclusions Overall, our results showcase that engineering endogenous plasma membrane ABC transporters is a promising approach for hydrophobic product efflux in S. cerevisiae. We also highlight the importance of improving cell physiology to enhance the efficiency of ABC transporters, especially energy status and cell membrane properties.

show abstract

Section: Mining Potential Transporters For β-Carotene Effluxsupporting

confidence: 76%

Section: Resultsmentioning

confidence: 99%

Section: Improving Membrane Fluidity To Enhance β-Carotene Secretionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Engineering endogenous ABC transporter with improving ATP supply and membrane flexibility enhances the secretion of β-carotene in Saccharomyces cerevisiae

Lin

Cheng

et al. 2020

Biotechnol Biofuels

View full text Add to dashboard Cite

show abstract

“…Directed evolution and rational protein design have been used to engineer enzymes in heterologous pathways (Eriksen et al, 2014;Niu et al, 2018;Hong et al, 2019; Supplementary Figure S1E). Monoterpenes are synthesized from geranyl diphosphate (GPP), which is also the precursor for the biosynthesis of FPP.…”

Section: Pathway Enzyme Designmentioning

confidence: 99%

Production of Terpenoids by Synthetic Biology Approaches

Zhang

Hong

2020

Front. Bioeng. Biotechnol.

View full text Add to dashboard Cite

Terpenoids are a large family of natural products with remarkable diverse biological functions, and have a wide range of applications as pharmaceuticals, flavors, pigments, and biofuels. Synthetic biology is presenting possibilities for sustainable and efficient production of high value-added terpenoids in engineered microbial cell factories, using Escherichia coli and Saccharomyces cerevisiae which are identified as well-known industrial workhorses. They also provide a promising alternative to produce non-native terpenes on account of available genetic tools in metabolic engineering and genome editing. In this review, we summarize the recent development in terpenoids production by synthetic biology approaches.

show abstract

Glucose 6‐phosphate dehydrogenase variants increase NADPH pools for yeast isoprenoid production

Adusumilli,

Alikkam Veetil,

Choudhury

et al. 2024

FEBS Open Bio

View full text Add to dashboard Cite

Isoprenoid biosynthesis has a significant requirement for the co‐factor NADPH. Thus, increasing NADPH levels for enhancing isoprenoid yields in synthetic biology is critical. Previous efforts have focused on diverting flux into the pentose phosphate pathway or overproducing enzymes that generate NADPH. In this study, we instead focused on increasing the efficiency of enzymes that generate NADPH. We first established a robust genetic screen that allowed us to screen improved variants. The pentose phosphate pathway enzyme, glucose 6‐phosphate dehydrogenase (G6PD), was chosen for further improvement. Different gene fusions of G6PD with the downstream enzyme in the pentose phosphate pathway, 6‐phosphogluconolactonase (6PGL), were created. The linker‐less G6PD‐6PGL fusion displayed the highest activity, and although it had slightly lower activity than the WT enzyme, the affinity for G6P was higher and showed higher yields of the diterpenoid sclareol in vivo. A second gene fusion approach was to fuse G6PD to truncated HMG‐CoA reductase, the rate‐limiting step and also the major NADPH consumer in the pathway. Both domains were functional, and the fusion also yielded higher sclareol levels. We simultaneously carried out a rational mutagenesis approach with G6PD, which led to the identification of two mutants of G6PD, N403D and S238QI239F, that showed 15–25% higher activity in vitro. The diterpene sclareol yields were also increased in the strains overexpressing these mutants relative to WT G6PD, and these will be very beneficial in synthetic biology applications.

show abstract

Efficient production of lycopene in Saccharomyces cerevisiae by enzyme engineering and increasing membrane flexibility and NAPDH production

Cited by 99 publications

References 39 publications

Engineering endogenous ABC transporter with improving ATP supply and membrane flexibility enhances the secretion of β-carotene in Saccharomyces cerevisiae

Engineering endogenous ABC transporter with improving ATP supply and membrane flexibility enhances the secretion of β-carotene in Saccharomyces cerevisiae

Production of Terpenoids by Synthetic Biology Approaches

Glucose 6‐phosphate dehydrogenase variants increase NADPH pools for yeast isoprenoid production

Contact Info

Product

Resources

About