Functional expression of eukaryotic cytochrome P450s in yeast

Jiang, Lihong; Huang, Lei; Cai, Jin; Xu, Zhinan; Lian, Jiazhang

doi:10.1002/bit.27630

Cited by 36 publications

(30 citation statements)

References 95 publications

(123 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Considering the significance in natural product biosynthesis as well as the low activity of P450s, various strategies have been attempted to address the challenges in functional expression of P450s in yeast. In contrast to previous strategies, such as N-terminal truncation and protein molecular engineering, the establishment of platform strains, especially those with ER expansion, has attracted more attention ( Jiang et al, 2021 ). As most eukaryotic P450s and CPRs are membrane-bound proteins and anchored to the ER outer membranes, ER expansion has been determined to be a generally applicable strategy to improve the functional expression of P450s Emmerstorfer et al found that the overexpression of ICE2 could increase the stability and activity of P450/CPR ( Emmerstorfer et al, 2015 ).…”

Section: Discussionmentioning

confidence: 99%

“…Although a variety of P450s have been successfully expressed in S. cerevisiae , low expression level and activity have become the biggest challenge for fundamental and biotechnological application studies of P450s ( Jiang et al, 2021 ). Accordingly, different strategies have been implemented to increase the expression level and/or activity of P450s in S. cerevisiae , including N-terminal truncation, protein molecular modification of P450s through protein engineering, and co-expression with CPRs ( Jiang et al, 2021 ). Unfortunately, the effects of these engineering strategies are often varied case by case.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Improved Functional Expression of Cytochrome P450s in Saccharomyces cerevisiae Through Screening a cDNA Library From Arabidopsis thaliana

Jiang

Dong

Liu

et al. 2021

Front. Bioeng. Biotechnol.

Self Cite

View full text Add to dashboard Cite

Cytochrome P450 enzymes (P450s) are a superfamily of heme-thiolate proteins widely existing in various organisms and play a key role in the metabolic network and secondary metabolism. However, the low expression levels and activities have become the biggest challenge for P450s studies. To improve the functional expression of P450s in Saccharomyces cerevisiae, an Arabidopsis thaliana cDNA library was expressed in the betaxanthin-producing yeast strain, which functioned as a biosensor for high throughput screening. Three new target genes AtGRP7, AtMSBP1, and AtCOL4 were identified to improve the functional expression of CYP76AD1 in yeast, with accordingly the accumulation of betaxanthin increased for 1.32-, 1.86-, and 1.10-fold, respectively. In addition, these three targets worked synergistically/additively to improve the production of betaxanthin, representing a total of 2.36-fold improvement when compared with the parent strain. More importantly, these genes were also determined to effectively increase the activity of another P450 enzyme (CYP736A167), catalyzing the hydroxylation of α-santalene to produce Z-α-santalol. Simultaneous overexpression of AtGRP7, AtMSBP1, and AtCOL4 increased α-santalene to Z-α-santalol conversion rate for more than 2.97-fold. The present study reported a novel strategy to improve the functional expression of P450s in S. cerevisiae and promises the construction of platform yeast strains for the production of natural products.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Improved Functional Expression of Cytochrome P450s in Saccharomyces cerevisiae Through Screening a cDNA Library From Arabidopsis thaliana

Jiang

Dong

Liu

et al. 2021

Front. Bioeng. Biotechnol.

Self Cite

View full text Add to dashboard Cite

show abstract

“…Compared with bacterial cell factories (i.e. Escherichia coli ), the ability of post-translational modifications and the presence of inner membrane systems make yeasts including P. pastoris preferred hosts to express eukaryotic complex proteins, such as cytochrome P450s (CYPs), which are often involved in the biosynthesis of natural products [ 6 ]. When compared with the model yeast Saccharomyces cerevisiae , P. pastoris has the advantage of strong and tightly regulated promoters for high level expression of recombinant proteins [ 7 ].…”

Section: Introductionmentioning

confidence: 99%

Development of synthetic biology tools to engineer Pichia pastoris as a chassis for the production of natural products

Gao

Jiang

Lian

2021

Synthetic and Systems Biotechnology

Self Cite

View full text Add to dashboard Cite

The methylotrophic yeast Pichia pastoris (a.k.a. Komagataella phaffii ) is one of the most commonly used hosts for industrial production of recombinant proteins. As a non-conventional yeast, P. pastoris has unique biological characteristics and its expression system has been well developed. With the advances in synthetic biology, more efforts have been devoted to developing P. pastoris into a chassis for the production of various high-value compounds, such as natural products. This review begins with the introduction of synthetic biology tools for the engineering of P. pastoris , including vectors, promoters, and terminators for heterologous gene expression as well as Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated System (CRISPR/Cas) for genome editing. This review is then followed by examples of the production of value-added natural products in metabolically engineered P. pastoris strains. Finally, challenges and outlooks in developing P. pastoris as a synthetic biology chassis are prospected.

show abstract

“…Due to the growing number of newly characterized CYP, many new heterologous candidates, which possibly have improved substrate preference and high activity in S. cerevisiae could be selected and compared to the ones used in Wernig et al, 2020a. Otherwise, a recent review (Jiang et al, 2020) summarized and discussed frequently applied strategies to improve the expression and performances of heterologous CYP in yeast. These include codon optimization, N-terminal modification, protein engineering, co-expression of CYP and CPR, fusion proteins of CYP-CPR and strain modifications to improve the microenvironment for CYP expression (Jiang et al, 2020). Codon optimization of heterologous genes is a wellestablished strategy that can improve gene expression (Elena et al, 2014).…”

Section: Saccharomyces Cerevisiaementioning

confidence: 99%

“…The activity of eukaryotic CYP requires efficient electron transfer from a CPR redox partner (Munro et al, 2013). A suitable CPR should be co-expressed because endogenous CPRs may not be compatible with the heterologous CYP (Jiang et al, 2020). For the reaction of OA to 8hydroxyoctanoic acid in Wernig et al, 2020a, efficient electron transfer to the CYP was ensured by expression of a cognate CPR.…”

Section: Saccharomyces Cerevisiaementioning

confidence: 99%

Metabolic engineering of Saccharomyces cerevisiae for production of medium-chain fatty acids and their derivatives

Wernig¹

View full text Add to dashboard Cite

The oleochemical and petrochemical industries provide diverse chemicals used in personal care products, food and pharmaceutical industries or as fuels, oils, polymers and others. However, fossil resources are dwindling and concerns about these conventional production methods have risen due to their strong negative impact on the environment and contribution to climate change. Therefore, alternative, sustainable and environmentally friendly production methods for oleochemical compounds such as fatty acids, fatty alcohols, hydroxy fatty acids and dicarboxylic acids are desired. The biotechnological production by engineered microorganism could fulfill these requirements. The concept of metabolic engineering, which is the modification of metabolic pathways of a host organism for increased production of a target compound, is a widely used strategy in biotechnology to generate cell factories or chassis strains for robust, efficient and high production. In this work, the versatile model and industrial yeast Saccharomyces cerevisiae was manipulated by metabolic engineering strategies for increased production of the medium-chain fatty acid octanoic acid and de novo production the derived 8-hydroxyoctanoic acid. Octanoic acid production was enabled by the fatty acid biosynthesis pathway by use of a mutated fatty acid synthase (FASRK) in a wild type FAS deficient strain. The yeast fatty acid synthase (FAS) consists of two polypeptides, α and β, which assemble to a α6β6 complex in a co-translational manner by interaction of the subunits. Because this step might be subject to cellular regulation, the α- and β- subunits of fatty acid synthase were fused to form a single-chain construct (fusFASRK), which displayed superior octanoic acid production compared with split FASRK. Thus, FASRK expression was identified as a limiting step of octanoic acid production. But the strains that produce octanoic acid have a severe growth defect that is undesirable for biotechnological applications and could lead to lower production titers. One reason is the strong inhibitory effect of octanoic acid. Another possibility is that the mutant FAS no longer produces enough essential long-chain fatty acids. To compensate for this, the mutated split and fused FAS variants were co-expressed individually in a strain harboring genomic wild type FAS alleles. In addition, mutant and wild type variants of fused and split FAS were co-expressed together in a FAS deficient strain. However, both cases resulted in decreased octanoic acid titers potentially by physical and/or metabolic crosstalk of the FAS variants. The fatty acid biosynthesis relies on cytosolic acetyl-CoA for initiation and derived malonyl-CoA for elongation and requires NADPH for reductive power. To increase production of octanoic acid, engineering strategies for increased acetyl-CoA and NADHP supply were investigated. First, the flux through the native cytosolic acetyl-CoA and NADPH providing pyruvate dehydrogenase bypass was enhanced by overexpression of the target genes ADH2, ALD6 and ACSL461P from Salmonella enterica in combination or individually. Next, the acety-CoA forming heterologous phosphoketolase/phosphotransacetylase pathway was expressed and NADPH formation was increased by redirecting the flux of glucose-6-phosphate into the NADPH producing oxidative branch of the pentose phosphate pathway. In particular, the flux through glycolysis and pyruvate dehydrogenase bypass was reduced by downregulating the expression of the phosphoglucose isomerase PGI1 and deleting the acetaldehyde dehydrogenase ALD6. Glucose-6-phosphate was guided into the pentose phosphate pathway by overexpressing the glucose-6-phosphate dehydrogenase ZWF1. The first approach did not influence octanoic acid production but the latter increased yields in the glucose consumption phase by 65 %. However, combining the superior fusFASRK with acetyl-CoA and NADPH supply engineering strategies did not result in additive production effects, indicating that other limitations hinder high octanoic acid accumulation. Limitations could be caused in particular by the strong inhibitory effects of octanoic acid or by intrinsic limitations of the FASRK mutant. To enlarge the octanoic acid production platform towards other derived valuable oleochemical compounds the de novo production of 8-hydroxyoctanoic acid was targeted. Since short- and medium-chain fatty acids have a strong inhibitory effect on Saccharomyces cerevisiae, the inhibitory effect of hydroxy fatty acid and dicarboxylic with eight or ten carbon atoms were compared and revealed only little or no growth impairment. Subsequently, the formation of 8-hydroxyoctanoic acid was targeted by a terminal hydroxylation of externally supplied octanoic acid in a bioconversion. For that, three heterologous genes, encoding for cytochromes P450 enzymes and their cognate cytochrome P450 reductases were expressed and 8-hydroxyoctanoic acid production was compared. In addition, the use of different carbon sources was compared. ...

show abstract

Functional expression of eukaryotic cytochrome P450s in yeast

Cited by 36 publications

References 95 publications

Improved Functional Expression of Cytochrome P450s in Saccharomyces cerevisiae Through Screening a cDNA Library From Arabidopsis thaliana

Improved Functional Expression of Cytochrome P450s in Saccharomyces cerevisiae Through Screening a cDNA Library From Arabidopsis thaliana

Development of synthetic biology tools to engineer Pichia pastoris as a chassis for the production of natural products

Metabolic engineering of Saccharomyces cerevisiae for production of medium-chain fatty acids and their derivatives

Contact Info

Product

Resources

About