Metabolic engineering of microbes for monoterpenoid production

Zhu, Kun Yan; Kong, Jing; Zhao, Baixiang; Rong, Lanxin; Liu, Shiqi; Lu, Zhihui; Zhang, Cuiying; Xiao, Dongguang; Pushpanathan, Krithi; Foo, Jee Loon; Wong, Adison; Yu, Aiqun

doi:10.1016/j.biotechadv.2021.107837

Cited by 25 publications

(19 citation statements)

References 147 publications

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“…Complemented by advances in gene synthesis, expression engineering and pathway discovery, biologists have refined metabolic engineering principles to develop microbial cell factories that can synthesize non-native, plant natural products [ 8 , 9 ]. Numerous terpenoid compounds, including limonene, bisabolol, isoprene and squalene, have been produced by fermentation technology [ 10 , 11 ]. While reviewing literatures on triterpenoid biosynthesis in yeasts, we came across two pioneering studies of enabling amyrin biosynthesis in the Baker’s yeast, Saccharomyces cereivisae .…”

Section: Introductionmentioning

confidence: 99%

Enhanced production of amyrin in Yarrowia lipolytica using a combinatorial protein and metabolic engineering approach

Kong

Ling

et al. 2022

Microb Cell Fact

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Background Amyrin is an important triterpenoid and precursor to a wide range of cosmetic, pharmaceutical and nutraceutical products. In this study, we metabolically engineered the oleaginous yeast, Yarrowia lipolytica to produce α- and β-amyrin on simple sugar and waste cooking oil. Results We first validated the in vivo enzymatic activity of a multi-functional amyrin synthase (CrMAS) from Catharanthus roseus, by expressing its codon-optimized gene in Y. lipolytica and assayed for amyrins. To increase yield, prevailing genes in the mevalonate pathway, namely HMG1, ERG20, ERG9 and ERG1, were overexpressed singly and in combination to direct flux towards amyrin biosynthesis. By means of a semi-rational protein engineering approach, we augmented the catalytic activity of CrMAS and attained ~ 10-folds higher production level on glucose. When applied together, protein engineering with enhanced precursor supplies resulted in more than 20-folds increase in total amyrins. We also investigated the effects of different fermentation conditions in flask cultures, including temperature, volumetric oxygen mass transfer coefficient and carbon source types. The optimized fermentation condition attained titers of at least 100 mg/L α-amyrin and 20 mg/L β-amyrin. Conclusions The design workflow demonstrated herein is simple and remarkably effective in amplifying triterpenoid biosynthesis in the yeast Y. lipolytica.

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Section: Introductionmentioning

confidence: 99%

Enhanced production of amyrin in Yarrowia lipolytica using a combinatorial protein and metabolic engineering approach

Kong

Ling

et al. 2022

Microb Cell Fact

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“…The MVA pathway has been proved to be an energy-consuming pathway with a significant demand for NAD(P)H and ATP . Specifically, two molecules of the universal electron carrier NADPH are needed by HMG-CoA reductase and three molecules of the energy currency ATP are required for MVA kinase, phosphomevalonate kinase, and phosphomevalonate decarboxylase to produce one molecule of IPP or DMAPP through the MVA pathway. , Of the various manipulation approaches, conferring yeast-altered organelle functional phenotypes has enabled efficient co-factor regulation, which could benefit subcellular pathways.…”

Section: Resultsmentioning

confidence: 99%

Mitochondrial Engineering of Yarrowia lipolytica for Sustainable Production of α-Bisabolene from Waste Cooking Oil

Zhu

Zhao

Zhang

et al. 2022

ACS Sustainable Chem. Eng.

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Metabolic engineering of yeasts for terpenoid production has mostly focused on the cytoplasm, whereas harnessing their organelles as subcellular factories has been overlooked. Herein, the farnesyl diphosphate synthetic pathway and α-bisabolene synthase were compartmentalized into the oleaginous yeast Yarrowia lipolytica's mitochondria to enable high-level α-bisabolene production. Through comprehensive metabolic engineering approaches, we exploited the potential and capability of the mitochondria as a subcellular factory to achieve 257.4 mg/L of α-bisabolene production from glucose. By combining mitochondrial and cytoplasmic engineering, we further boosted the α-bisabolene titer to 765.1 mg/L by utilizing waste cooking oil as the sole carbon source. Finally, the α-bisabolene titer of the resulting strain reached 1058.1 mg/L in a 5 L bioreactor, which is the highest titer in the engineered Y. lipolytica cell factory reported to date. Overall, our study has provided valuable insights into the mitochondrial engineering of Y. lipolytica for sustainable and green production of valuable compounds.

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“…In Y. lipolytica, this process produces acetyl-CoA which then enters the glyoxylate cycle for synthesis of the IA precursor, cis-aconitic acid ( Dominguez et al, 2010 ; Koivistoinen et al, 2013 ; Xu et al, 2017 ). Several studies have shown that subcellular localization of specific enzymes or metabolic pathways not only increase product conversion efficiency, but is also able to suppress the undesirable effects of competitive metabolic inhibition ( Zhu et al, 2018 ; Yang et al, 2019 ; Zhu et al, 2021 ). As such, this approach of subcellular compartmentalization is adopted in our study and complemented with the use of WCO as the substrate to enable sustainable, efficient and low-cost production of IA.…”

Section: Resultsmentioning

confidence: 99%

Engineering Yarrowia lipolytica to Produce Itaconic Acid From Waste Cooking Oil

Rong

Ling

Wang

et al. 2022

Front. Bioeng. Biotechnol.

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Itaconic acid (IA) is a high-value organic acid with a plethora of industrial applications. In this study, we seek to develop a microbial cell factory that could utilize waste cooking oil (WCO) as raw material for circular and cost-effective production of the abovementioned biochemical. Specifically, we expressed cis-aconitic acid decarboxylase (CAD) gene from Aspergillus terreus in either the cytosol or peroxisome of Yarrowia lipolytica and assayed for production of IA on WCO. To further improve production yield, the 10 genes involved in the production pathway of acetyl-CoA, an intermediate metabolite necessary for the synthesis of cis-aconitic acid, were individually overexpressed and investigated for their impact on IA production. To minimize off-target flux channeling, we had also knocked out genes related to competing pathways in the peroxisome. Impressively, IA titer up to 54.55 g/L was achieved in our engineered Y. lipolytica in a 5 L bioreactor using WCO as the sole carbon source.

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Metabolic engineering of microbes for monoterpenoid production

Cited by 25 publications

References 147 publications

Enhanced production of amyrin in Yarrowia lipolytica using a combinatorial protein and metabolic engineering approach

Enhanced production of amyrin in Yarrowia lipolytica using a combinatorial protein and metabolic engineering approach

Mitochondrial Engineering of Yarrowia lipolytica for Sustainable Production of α-Bisabolene from Waste Cooking Oil

Engineering Yarrowia lipolytica to Produce Itaconic Acid From Waste Cooking Oil

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