Engineering promiscuity of chloramphenicol acetyltransferase for microbial designer ester biosynthesis

Seo, Hyung-Min; Lee, Jun Young; Giannone, Richard J.; Dunlap, Noah J.; Trinh, Cong T.

doi:10.1016/j.ymben.2021.04.005

Cited by 27 publications

(24 citation statements)

References 60 publications

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“…Although our study focused on the selective microbial biosynthesis of isoamyl acetate as a proof-of-concept, the generalizable modular design of the recursive one-carbon elongation pathway can be extended to produce longer branched-chain acetate esters such as isohexyl acetate and isoheptyl acetate. Since CATec3 Y20F has higher catalytic efficiency towards longer chain alcohols (Seo et al, 2021), it is expected that the selectivity of KDC and/or LeuA needs to be further engineered to achieve selective microbial biosynthesis of designer acetate esters with longer carbon chain lengths.…”

Section: Discussionmentioning

confidence: 99%

“…1 μL sample was injected into the column with the 1:50 split mode at an injector temperature of 280°C. For the MS system, selected ion mode (SIM) was used to detect and quantify esters with the parameters described previously(Seo et al, 2021). As an internal standard, 10 mg/L n-decane were added in initial hexadecane layer and detected with m/z 85, 99, and 113 from 12 to 15 m retention time range.…”

Section: Methodsmentioning

confidence: 99%

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Proteome reallocation enables the selective de novo biosynthesis of non-linear, branched-chain acetate esters

Seo

Giannone

Yang

et al. 2022

Preprint

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The one-carbon recursive ketoacid elongation pathway is responsible for making various branched-chain amino acids, aldehydes, alcohols, and acetate esters in living cells. Controlling selective microbial biosynthesis of these target molecules at high efficiency is challenging due to enzyme promiscuity, regulation, and metabolic burden. In this study, we present a systematic modular design approach to control proteome reallocation for selective microbial biosynthesis of branched-chain acetate esters. Through pathway modularization, we partitioned the branched-chain ester pathways into four submodules including keto-isovalerate submodule for converting pyruvate to keto-isovalerate, ketoacid elongation submodule for producing longer carbon-chain keto-acids, ketoacid decarboxylase submodule for converting ketoacids to alcohols, and alcohol acyltransferase submodule for producing branched-chain acetate esters by condensing alcohols and acetyl-CoA. By systematic manipulation of pathway gene replication and transcription, enzyme specificity of the first committed steps of these submodules, and downstream competing pathways, we demonstrated selective microbial production of isoamyl acetate over isobutyl acetate. We found that the optimized isoamyl acetate pathway globally redistributed the amino acid fractions in the proteomes and required up to 23-31% proteome reallocation at the expense of other cellular resources, such as those required to generate precursor metabolites and energy for growth and amino acid biosynthesis. The engineered strains produced isoamyl acetate at a titer of 8.8 g/L (> 0.25 g/L toxicity limit), a yield of 0.17 g/g (47% of maximal theoretical value), and 86% selectivity, achieving the highest titers, yields and selectivity of isoamyl acetate reported to date.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Proteome reallocation enables the selective de novo biosynthesis of non-linear, branched-chain acetate esters

Seo

Giannone

Yang

et al. 2022

Preprint

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Section: Cellularly-mediated Transformationsmentioning

confidence: 99%

“…Indeed, this detoxification strategy has been successfully exploited for microbial biosynthesis of shorter chain esters at high levels exceeding their solubility limit and inhibition threshold. 171 On the other hand, continuous product removal appears to be challenging to implement in the presence of solid cellulosic feedstocks (see Section II.c).…”

Section: Cellularly-mediated Transformationsmentioning

confidence: 99%

“…159 Thermostable heterologous pathways can be now engineered in this organism to produce a broad range of fuel molecules such as isobutanol, 169 n-butanol 170 and a wide range of ester molecules including isobutyl acetate, n-butyl acetate, n-pentyl acetate, isoamyl acetate, geranyl acetate, and isobutyl isobutyrate. [171][172][173] Table 4 lists some representative commodity products, the host organisms used for commercial production, and deciding factors underlying the choice of host organism.…”

Section: Reviewmentioning

confidence: 99%

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Toward low-cost biological and hybrid biological/catalytic conversion of cellulosic biomass to fuels

Lynd

Beckham

Guss

et al. 2022

Energy Environ. Sci.

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104

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Probing specificities of alcohol acyltransferases for designer ester biosynthesis with a high‐throughput microbial screening platform

Lee

Seo

Young

et al. 2021

Biotech & Bioengineering

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Alcohol acyltransferases (AATs) enables microbial biosynthesis of a large space of esters by condensing an alcohol and an acyl-CoA. However, substrate promiscuity of AATs prevents microbial biosynthesis of designer esters with high selectivity. Here, we developed a high-throughput microbial screening platform that facilitates rapid identification of AATs for designer ester biosynthesis. First, we established a microplate-based culturing technique with in situ fermentation and extraction of esters. We validated its capability in rapid profiling of the alcohol substrate specificity of 20 chloramphenicol acetyltransferase variants derived from Staphylococcus aureus (CAT Sa ) for microbial biosynthesis of acetate esters with various exogeneous alcohol supply. By coupling the microplate-based culturing technique with a previously established colorimetric assay, we developed a high-throughput microbial screening platform for AATs. We demonstrated that this platform could not only probe the alcohol substrate specificity of both native and engineered AATs but also identify the beneficial mutations in engineered AATs for enhanced ester synthesis.We anticipate the high-throughput microbial screening platform provides a useful tool to identify novel wildtype and engineered AATs that have important roles in nature and industrial biocatalysis for designer bioester production.

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Engineering promiscuity of chloramphenicol acetyltransferase for microbial designer ester biosynthesis

Cited by 27 publications

References 60 publications

Proteome reallocation enables the selective de novo biosynthesis of non-linear, branched-chain acetate esters

Proteome reallocation enables the selective de novo biosynthesis of non-linear, branched-chain acetate esters

Toward low-cost biological and hybrid biological/catalytic conversion of cellulosic biomass to fuels

Probing specificities of alcohol acyltransferases for designer ester biosynthesis with a high‐throughput microbial screening platform

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