Production of Supply-limited Natural Product Therapeutics Using Engineered Yeast

Jay D KEASLING 한국생물공학회 2023 한국생물공학회 학술대회 Vol.2023 No.10

Engineered Polyketide Synthases as Platform for Synthetic Chemistry

Jay D. KEASLING 한국생물공학회 2021 한국생물공학회 학술대회 Vol.2021 No.10

Polyketides are one of the largest classes of natural products, possessing immense structural diversity and complex chemical architectures. Many polyketides (PKs) are among the most important secondary metabolites for their applications in medicine, agriculture, and industry. Examples include anticancer drugs (epothilone), antibiotics (erythromycin), insecticides (spinosyn A) and antifungals (amphotericin B). These particular examples of polyketides are biosynthesized by multimodular enzyme complexes known as type I modular polyketide synthases (PKSs). Working in an assembly-line fashion, multimodular PKSs assemble and tailor readily available acyl-CoAs within the host cell into large, complex, chiral molecules. Each of these PKSs comprises a series of modules that can be further dissected into a series of domains responsible for the extension of the polyketide back- bone through condensation and selective reductive processing of an acyl-CoA building block. The collinear architecture of these modules, apparent by inspection of the domains present and the predictive selectivity motifs harbored within, provide insights into the chemical connectivity and stereochemical configuration of the polyketide metabolite from analysis of its coding sequence. While PKSs have been traditionally studied for the production of pharmaceuticals, engineered modular PKSs have the potential to be an extraordinarily effective retrosynthesis platform for the bio-production of products from biofuels and commodity chemicals to both pharmaceutical and nonpharmaceutical fine and specialty chemicals. By rearranging existing polyketide modules and domains, one can exquisitely control chemical structure from DNA sequence alone. However, this potential has only just begun to be realized as the compounds that have been made using engineered PKSs represent a small fraction of the potentially accessible chemical space. In my talk, I will highlight work from our laboratory where we have engineered PKSs to produce a variety of commodity and specialty chemicals and developed software and high throughput robotic platforms to enable design and construction of PKSs in high throughput.

Intracellular cellobiose metabolism and its applications in lignocellulose-based biorefineries

Parisutham, Vinuselvi,Chandran, Sathesh-Prabu,Mukhopadhyay, Aindrila,Lee, Sung Kuk,Keasling, Jay D. Elsevier Applied Science 2017 Bioresource technology Vol.239 No.-

<P><B>Abstract</B></P> <P>Complete hydrolysis of cellulose has been a key characteristic of biomass technology because of the limitation of industrial production hosts to use cellodextrin, the partial hydrolysis product of cellulose. Cellobiose, a <I>β</I>-1,4-linked glucose dimer, is a major cellodextrin of the enzymatic hydrolysis (via endoglucanase and exoglucanase) of cellulose. Conversion of cellobiose to glucose is executed by <I>β</I>-glucosidase. The complete extracellular hydrolysis of celluloses has several critical barriers in biomass technology. An alternative bioengineering strategy to make the bioprocessing less challenging is to engineer microbes with the abilities to hydrolyze and assimilate the cellulosic-hydrolysate cellodextrin. Microorganisms engineered to metabolize cellobiose rather than the monomeric glucose can provide several advantages for lignocellulose-based biorefineries. This review describes the recent advances and challenges in engineering efficient intracellular cellobiose metabolism in industrial hosts. This review also describes the limitations of and future prospectives in engineering intracellular cellobiose metabolism.</P> <P><B>Highlights</B></P> <P> <UL> <LI> The complete hydrolysis of cellulose by cellulase cocktail poses several critical barriers. </LI> <LI> The intracellular cellobiose assimilation has been considered as an alternative strategy. </LI> <LI> Engineering the industrial hosts for efficient cellobiose utilization would be advantageous. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Autonomous control of metabolic state by a quorum sensing (QS)-mediated regulator for bisabolene production in engineered <i>E. coli</i>

Kim, Eun-Mi,Woo, Han Min,Tian, Tian,Yilmaz, Suzan,Javidpour, Pouya,Keasling, Jay D.,Lee, Taek Soon Academic Press 2017 Metabolic engineering Vol.44 No.-

<P><B>Abstract</B></P> <P>Inducible gene expression systems are widely used in microbial host strains for protein and commodity chemical production because of their extensive characterization and ease of use. However, some of these systems have disadvantages such as leaky expression, lack of dynamic control, and the prohibitively high costs of inducers associated with large-scale production. Quorum sensing (QS) systems in bacteria control gene expression in response to population density, and the LuxI/R system from <I>Vibrio fischeri</I> is a well-studied example. A QS system could be ideal for biofuel production strains as it is self-regulated and does not require the addition of inducer compounds, which reduce operational costs for inducer. In this study, a QS system was developed for inducer-free production of the biofuel compound bisabolene from engineered <I>E. coli</I>. Seven variants of the Sensor plasmid, which carry the <I>luxI</I>-<I>luxR</I> genes, and four variants of the Response plasmid, which carry bisabolene producing pathway genes under the control of the P<SUB> <I>luxI</I> </SUB> promoter, were designed for optimization of bisabolene production. Furthermore, a chromosome-integrated QS strain was engineered with the best combination of Sensor and Response plasmid and produced bisabolene at a titer of 1.1g/L without addition of external inducers. This is a 44% improvement from our previous inducible system. The QS strain also displayed higher homogeneity in gene expression and isoprenoid production compared to an inducible-system strain.</P> <P><B>Highlights</B></P> <P> <UL> <LI> A quorum-sensing host was developed for inducer-free biofuel production in <I>E. coli</I>. </LI> <LI> Systematic engineering of the quorum-sensing system generated efficient QS hosts. </LI> <LI> Chromosomal integration of QS system generated a versatile inducer-free platform. </LI> <LI> The QS integrated strain produced a biofuel at higher yields than inducible system. </LI> <LI> QS system confirms higher homogeneity in gene expression and isoprenoid production. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Technical Advances to Accelerate Modular Type I Polyketide Synthase Engineering towards a Retro-biosynthetic Platform

Bo Pang,Luis E. Valencia,Jessica Wang,Yao Wan,Ravi Lal,Amin Zargar,Jay D. Keasling 한국생물공학회 2019 Biotechnology and Bioprocess Engineering Vol.24 No.3

Modular type I polyketide synthases (PKSs) are multifunctional proteins that are comprised of individual domains organized into modules. These modules act together to assemble complex polyketides from acyl-CoA substrates in a linear fashion. This assembly-line enzymology makes engineered PKSs a potential retrobiosynthetic platform to produce fuels, commodity chemicals, speciality chemicals, and pharmaceuticals in various host microorganisms, including bacteria and fungi. However, the realization of this potential is restricted by practical difficulties in strain engineering, protein overexpression, and titer/yield optimization. These challenges are becoming more possible to overcome due to technical advances in PKS design, engineered heterologous hosts, DNA synthesis and assembly, PKS heterologous expression, and analytical methodology. In this review, we highlight these technical advances in PKS engineering and provide practical considerations thereof.

Farnesol production from <i>Escherichia coli</i> by harnessing the exogenous mevalonate pathway

Wang, Chonglong,Yoon, Sang‐,Hwal,Shah, Asad Ali,Chung, Young‐,Ryun,Kim, Jae‐,Yean,Choi, Eui‐,Sung,Keasling, Jay D.,Kim, Seon‐,Won Wiley Subscription Services, Inc., A Wiley Company 2010 Biotechnology and bioengineering Vol.107 No.3

<P><B>Abstract</B></P><P>Farnesol (FOH) production has been carried out in metabolically engineered <I>Escherichia coli</I>. FOH is formed through the depyrophosphorylation of farnesyl pyrophosphate (FPP), which is synthesized from isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) by FPP synthase. In order to increase FPP synthesis, <I>E. coli</I> was metabolically engineered to overexpress <I>ispA</I> and to utilize the foreign mevalonate (MVA) pathway for the efficient synthesis of IPP and DMAPP. Two‐phase culture using a decane overlay of the culture broth was applied to reduce volatile loss of FOH produced during culture and to extract FOH from the culture broth. A FOH production of 135.5 mg/L was obtained from the recombinant <I>E. coli</I> harboring the pTispA and pSNA plasmids for <I>ispA</I> overexpression and MVA pathway utilization, respectively. It is interesting to observe that a large amount of FOH could be produced from <I>E. coli</I> without FOH synthase by the augmentation of FPP synthesis. Introduction of the exogenous MVA pathway enabled the dramatic production of FOH by <I>E. coli</I> while no detectable FOH production was observed in the endogenous MEP pathway‐only control. Biotechnol. Bioeng. 2010;107: 421–429. © 2010 Wiley Periodicals, Inc.</P>

Identification of Genes Affecting Lycopene Accumulation in Escherichia coli Using a Shot-Gun Method

Kang, Min-Jung,Lee, Young-Mi,Yoon, Sang-Hwal,Kim, Jung-Heon,Ock, So-Won,Jung, Kyung-Hwa,Shin, Yong-Chul,Jay D. Keasling,Kim, Seon-Won Plant molecular biology and biotechnology research 2005 Plant molecular biology and biotechnology research Vol.2005 No.

Genes enhancing lycopene production in Escherichia coli were identified through colorimetric screening of shot-gun library clones constructed with E. coli chromosomal DNA. These E. coli cells had been engineered to produce lycopene, a red-colored carotenoid, which enabled screening for genes that enhance lycopene production. Six clones with enhanced lycopene production were isolated. Among 13 genes in these clones, dxs, appY,crl, and rpoSwere found to be involved in enhanced lycopene production. While dxs and rpoS have been already reported to enhance lycopene production, appY and crl have not. DXP (1-deoxy-D-xylulose-5-phosphate) synthase is encoded by dxs and participates in the rate-limiting step in the synthesis of isopentenyl pyrophosphate (IPP), a building block of lycopene. Sigma S factor, encoded by rpoS, regulates transcription of genes induced at the stationary phase. The appY and crl genes encode transcriptional regulators related to anaerobic energy metabolism and the formation of curli surface fibers, respectively. E. coli harboring appY plasmids produced 2.8 mg lycopene/g dry cell weight (DCW), the same amount obtained with dxs despite the fact that appY is not directly involved in the lycopene synthesis pathway, The co-expression of appY, crl, and rpoS with dxs synergistically enhanced lycopene production. The co-expression of appY with dxs produced eight times the amount of lycopene (4.7 mg/g DCW) that was produced without expression of both genes (0.6 mg/g DCW).

Identification of genes affecting lycopene accumulation in Escherichia coli using a shot-gun method

Kang, Min Jung,Lee, Young Mi,Yoon, Sang Hwal,Kim, Jung Heon,Ock, So Won,Jung, Kyung Hwa,Shin, Yong Chul,Keasling, Jay D.,Kim, Seon Won Wiley Subscription Services, Inc., A Wiley Company 2005 Biotechnology and bioengineering Vol.91 No.5

<P>Genes enhancing lycopene production in Escherichia coli were identified through colorimetric screening of shot-gun library clones constructed with E. coli chromosomal DNA. These E. coli cells had been engineered to produce lycopene, a red-colored carotenoid, which enabled screening for genes that enhance lycopene production. Six clones with enhanced lycopene production were isolated. Among 13 genes in these clones, dxs, appY, crl, and rpoS were found to be involved in enhanced lycopene production. While dxs and rpoS have been already reported to enhance lycopene production, appY and crl have not. DXP (1-deoxy-D-xylulose-5-phosphate) synthase is encoded by dxs and participates in the rate-limiting step in the synthesis of isopentenyl pyrophosphate (IPP), a building block of lycopene. Sigma S factor, encoded by rpoS, regulates transcription of genes induced at the stationary phase. The appY and crl genes encode transcriptional regulators related to anaerobic energy metabolism and the formation of curli surface fibers, respectively. E. coli harboring appY plasmids produced 2.8 mg lycopene/g dry cell weight (DCW), the same amount obtained with dxs despite the fact that appY is not directly involved in the lycopene synthesis pathway. The co-expression of appY, crl, and rpoS with dxs synergistically enhanced lycopene production. The co-expression of appY with dxs produced eight times the amount of lycopene (4.7 mg/g DCW) that was produced without expression of both genes (0.6 mg/g DCW). © 2005 Wiley Periodicals, Inc.</P>

Enhanced lycopene production in Escherichia coli engineered to synthesize isopentenyl diphosphate and dimethylallyl diphosphate from mevalonate

Yoon, Sang-Hwal,Lee, Young-Mi,Kim, Ju-Eun,Lee, Sook-Hee,Lee, Joo-Hee,Kim, Jae-Yean,Jung, Kyung-Hwa,Shin, Yong-Chul,Keasling, Jay D.,Kim, Seon-Won Wiley Subscription Services, Inc., A Wiley Company 2006 Biotechnology and bioengineering Vol.94 No.6

<P>To increase expression of lycopene synthetic genes crtE, crtB, crtI, and ipiHP1, the four exogenous genes were cloned into a high copy pTrc99A vector with a strong trc promoter. Recombinant Escherichia coli harboring pT-LYCm4 produced 17 mg/L of lycopene. The mevalonate lower pathway, composed of mvaK1, mvaK2, mvaD, and idi, was engineered to produce pSSN12Didi for an efficient supply of the lycopene building blocks, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Mevalonate was supplied as a substrate for the mevalonate lower pathway. Lycopene production in E. coli harboring pT-LYCm4 and pSSN12Didi with supplementation of 3.3 mM mevalonate was more than threefold greater than bacteria with pT-LYCm4 only. Lycopene production was dependent on mevalonate concentration supplied in the culture. Clump formation was observed as cells accumulated more lycopene. Further clumping was prevented by adding the surfactant Tween 80 0.5% (w/v), which also increased lycopene production and cell growth. When recombinant E. coli harboring pT-LYCm4 and pSSN12Didi was cultivated in 2YT medium containing 2% (w/v) glycerol as a carbon source, 6.6 mM mevalonate for the mevalonate lower pathway, and 0.5% (w/v) Tween 80 to prevent clump formation, lycopene production was 102 mg/L and 22 mg/g dry cell weight, and cell growth had an OD<SUB>600</SUB> value of 15 for 72 h. © 2006 Wiley Periodicals, Inc.</P>