Microbial electrosynthesis from carbon dioxide feedstock linked to yeast growth for the production of high-value isoprenoids

Yadav, Ravineet; Chattopadhyay, Banani; Kiran, Rashmi; Yadav, Ankit; Bachhawat, Anand K.; Patil, Sunil A.

doi:10.1016/j.biortech.2022.127906

Cited by 7 publications

(3 citation statements)

References 39 publications

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“…The various mutants of ZWF1 were constructed by splice overlap extension PCR (PCR1 with Sc G6PD NheI‐FP and corresponding mutant RP; PCR2 with corresponding mutant FP and Sc G6PD XhoI‐RP; the third PCR used PCR1 and PCR2 products as templates with Sc G6PD NheI‐FP and Sc G6PD XhoI‐RP primers) and cloned into pRS313TEF and PET23a vectors. The sclareol biosynthetic genes copal‐8‐ol diphosphate synthase ( CcCLS ) and sclareol synthase ( SsSS ) that were custom synthesized [ 13 ] were sub cloned into yeast centromeric vectors pRS314TEF and p416TEF, respectively, to make them compatible for a four‐plasmid yeast transformation system. All the clones constructed were confirmed by sequencing.…”

Section: Methodsmentioning

confidence: 99%

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

Adusumilli,

Alikkam Veetil,

Choudhury

et al. 2024

FEBS Open Bio

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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.

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Section: Methodsmentioning

confidence: 99%

“…The sclareol peak was observed at the retention time of 13.25 min. Targeted peaks were identified by analyzing the mass spectra with the available NIST library and literature as described earlier [ 13 ]. The extracted ion chromatograms with ions of interest were analyzed (sclareol shows characteristic mass fragments of 177 191).…”

Section: Methodsmentioning

confidence: 99%

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

Adusumilli,

Alikkam Veetil,

Choudhury

et al. 2024

FEBS Open Bio

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“…In general, microorganisms produce sesquiterpenes from renewable carbon sources through intrinsic pathways, in which the precursor farnesyl pyrophosphate (FPP) is converted into molecules with diverse structures under the catalysis of specific sesquiterpene synthases (Liu et al, 2022; Yadav et al, 2022; Zhang, Wang, et al, 2021). However, β‐elemene is not the direct product of a sesquiterpene synthase, instead being derived from the natural product germacrene A via an intramolecular Cope rearrangement reaction under heating (Goore et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

Combination of microbial and chemical synthesis for the sustainable production of β‐elemene, a promising plant‐extracted anticancer compound

Li,

Mai,

Lin

et al. 2023

Biotech & Bioengineering

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Beta‐elemene, a class of sesquiterpene derived from the Chinese medicinal herb Curcuma wenyujin, is widely used in clinical medicine due to its broad‐spectrum antitumor activity. However, the unsustainable plant extraction prompted the search for environmentally friendly strategies for β‐elemene production. In this study, we designed a Yarrowia lipolytica cell factory that can continuously produce germacrene A, which is further converted into β‐elemene with 100% yield through a Cope rearrangement reaction by shifting the temperature to 250°C. First, the productivity of four plant‐derived germacrene A synthases was evaluated. After that, the metabolic flux of the precursor to germacrene A was maximized by optimizing the endogenous mevalonate pathway, inhibiting the competing squalene pathway, and expressing germacrene A synthase gene in multiple copies. Finally, the most promising strain achieved the highest β‐elemene titer reported to date with 5.08 g/L. This sustainable and green method has the potential for industrial β‐elemene production.

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Halophilic CO2-fixing microbial community as biocatalyst improves the energy efficiency of the microbial electrosynthesis process

Kiran

Yadav

Sathe

et al. 2023

Bioresource Technology

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Microbial electrosynthesis from carbon dioxide feedstock linked to yeast growth for the production of high-value isoprenoids

Cited by 7 publications

References 39 publications

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

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

Combination of microbial and chemical synthesis for the sustainable production of β‐elemene, a promising plant‐extracted anticancer compound

Halophilic CO2-fixing microbial community as biocatalyst improves the energy efficiency of the microbial electrosynthesis process

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