ORCID Profile
0000-0001-6918-6016
Current Organisations
University of Queensland
,
Ngee Ann Polytechnic
,
Queensland University of Technology
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Publisher: Cold Spring Harbor Laboratory
Date: 25-11-2022
DOI: 10.1101/2022.11.24.517869
Abstract: Enzyme spatial organisation and compartmentalisation are naturally evolved mechanisms for facilitating multi-step biocatalysis. We explored the synthetic in vivo co-encapsulation of two different cargo proteins in yeast using a self-assembling virus-like particle. Co-encapsulation was verified using single particle techniques for both end-to-end fusion of the cargo proteins with the encapsulation anchor at one end, and coexpression of each cargo protein with their in idual anchors. The co-encapsulation of a bifunctional geranyl diphosphate/farnesyl diphosphate synthase and a bifunctional linalool/nerolidol synthase delivered nerolidol titres up to 30 times that of an unorganised ‘free’ enzyme control, a remarkable improvement from a single engineering step. Interestingly, striking differences in the ratio of products (linalool and nerolidol) were observed with each spatial organisation approach. This work presents the largest reported titre fold increases from in vivo enzyme compartmentalisation and suggests that enzyme spatial organisation could be used to modulate the product profile of promiscuous enzymes.
Publisher: Wiley
Date: 26-09-2023
Publisher: Springer Science and Business Media LLC
Date: 24-05-2022
DOI: 10.1038/S41467-022-30529-8
Abstract: Bottlenecks in metabolic pathways due to insufficient gene expression levels remain a significant problem for industrial bioproduction using microbial cell factories. Increasing gene dosage can overcome these bottlenecks, but current approaches suffer from numerous drawbacks. Here, we describe HapAmp, a method that uses haploinsufficiency as evolutionary force to drive in vivo gene lification. HapAmp enables efficient, titratable, and stable integration of heterologous gene copies, delivering up to 47 copies onto the yeast genome. The method is exemplified in metabolic engineering to significantly improve production of the sesquiterpene nerolidol, the monoterpene limonene, and the tetraterpene lycopene. Limonene titre is improved by 20-fold in a single engineering step, delivering ∼1 g L −1 in the flask cultivation. We also show a significant increase in heterologous protein production in yeast. HapAmp is an efficient approach to unlock metabolic bottlenecks rapidly for development of microbial cell factories.
Publisher: Cold Spring Harbor Laboratory
Date: 09-11-2022
DOI: 10.1101/2022.11.08.515726
Abstract: The end-to-end fusion of enzymes that catalyse successive steps in a reaction pathway is a metabolic engineering strategy that has been successfully applied in a variety of pathways and is particularly common in terpene bioproduction. Despite its popularity, limited work has been done to interrogate the mechanism of metabolic enhancement from enzyme fusion. We observed a remarkable -fold improvement in nerolidol production upon translational fusion of nerolidol synthase (a sesquiterpene synthase) to farnesyl diphosphate synthase. This delivered a titre increase from 29.6 mg/L up to 4.2 g/L nerolidol in a single engineering step. Whole-cell proteomic analysis revealed that nerolidol synthase levels in the fusion strains were greatly elevated compared to the non-fusion control. Similarly, the fusion of nerolidol synthase to non-catalytic domains also produced comparable increases in titre, which coincided with improved enzyme expression. When farnesyl diphosphate synthase was fused to other terpene synthases, we observed more modest improvements in terpene titre (1.9- and 3.8-fold), which corresponds to increases of a similar magnitude in terpene synthase expression. Therefore, increased in vivo enzyme levels – resulting from improved expression and/or stability – is likely to be a major driver of catalytic enhancement from enzyme fusion.
Publisher: Wiley
Date: 09-09-2021
Abstract: The yeast Saccharomyces cerevisiae uses the pyruvate dehydrogenase‐bypass for acetyl‐CoA biosynthesis. This relatively inefficient pathway limits production potential for acetyl‐CoA‐derived biochemical due to carbon loss and the cost of two high‐energy phosphate bonds per molecule of acetyl‐CoA. Here, we attempted to improve acetyl‐CoA production efficiency by introducing heterologous acetylating aldehyde dehydrogenase and phosphoketolase pathways for acetyl‐CoA synthesis to enhance production of the sesquiterpene trans ‐nerolidol. In addition, we introduced auxin‐mediated degradation of the glucose‐dependent repressor Mig1p to allow induced expression of GAL promoters on glucose so that production potential on glucose could be examined. The novel genes that we used to reconstruct the heterologous acetyl‐CoA pathways did not sufficiently complement the loss of endogenous acetyl‐CoA pathways, indicating that superior heterologous enzymes are necessary to establish fully functional synthetic acetyl‐CoA pathways and properly explore their potential for nerolidol synthesis. Notwithstanding this, nerolidol production was improved twofold to a titre of ˜ 900 mg l −1 in flask cultivation using a combination of heterologous acetyl‐CoA pathways and Mig1p degradation. Conditional Mig1p depletion is presented as a valuable strategy to improve the productivities in the strains engineered with GAL promoters‐controlled pathways when growing on glucose.
Publisher: American Chemical Society (ACS)
Date: 23-01-2018
DOI: 10.1021/ACSSYNBIO.7B00355
Abstract: The GAL promoters are applied in metabolic engineering and synthetic biology to control gene expression in the budding yeast Saccharomyces cerevisiae. In gal80Δ background strains, they show diauxie-inducible expression, a feature beneficial in metabolic pathway optimization. However, only a limited number of GAL promoters have been characterized and are available for engineering purposes. Multiple uses of the same promoters can result in genetic instability in engineered strains due to homologous recombination. Here, 11 GAL1/2 promoters from other Saccharomyces species were isolated and characterized in S. cerevisiae. They exhibited diauxie-inducible expression patterns with low strength in exponential growth phase and induction in the ethanol growth phase. These promoters represent an expansion to the collection of GAL promoters available for genetic engineering in S. cerevisiae, including an increased ersity of expression levels. This provides the capacity for increased numbers of genetic manipulations with a lower risk of genetic instability.
Publisher: Springer Science and Business Media LLC
Date: 16-02-2022
DOI: 10.1038/S42003-022-03070-Z
Abstract: Temporal control of heterologous pathway expression is critical to achieve optimal efficiency in microbial metabolic engineering. The broadly-used GAL promoter system for engineered yeast ( Saccharomyces cerevisiae ) suffers from several drawbacks specifically, unintended induction during laboratory development, and unintended repression in industrial production applications, which decreases overall production capacity. Eukaryotic synthetic circuits have not been well examined to address these problems. Here, we explore a modularised engineering method to deploy new genetic circuits applicable for expanding the control of GAL promoter-driven heterologous pathways in S. cerevisiae . Trans - and cis - modules, including eukaryotic trans -activating-and-repressing mechanisms, were characterised to provide new and better tools for circuit design. A eukaryote-like tetracycline-mediated circuit that delivers stringent repression was engineered to minimise metabolic burden during strain development and maintenance. This was combined with a novel 37 °C induction circuit to relief glucose-mediated repression on the GAL promoter during the bioprocess. This delivered a 44% increase in production of the terpenoid nerolidol, to 2.54 g L −1 in flask cultivation. These negative ositive transcriptional regulatory circuits expand global strategies of metabolic control to facilitate laboratory maintenance and for industry applications.
Publisher: Elsevier BV
Date: 09-2023
No related grants have been discovered for Bingyin Peng.