Data is the average of three independent biological replicates, and error bars indicate standard error. Cells were serially diluted tenfold onto solid LB agar media with or without 0. Error bars are not plotted when they are shorter than the symbol used on the graph. To examine the tolerance of C.
We confirmed that E. In contrast, C. Wild-type C. Isopentenol evaporation was dependent on the cultivation format and was more pronounced in a well deep well plate format Additional file 1 D. These results indicated that C. Together, these results suggested that selecting C. Next, we expressed a heterologous mevalonate-based isopentenol biosynthesis pathway [ 28 ] in C.
Acetyl-coA is converted to mevalonate-phosphate by four enzymatic reactions, which in turn is decarboxylated to isopentenyl monophosphate a promiscuous activity from PMD.
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Isopentenyl monophosphate is then spontaneously dephosphorylated by an endogenous phosphatase to yield isopentenol Fig. To identify the cultivation conditions for optimal production of isopentenol in C. The d -glucose concentration was an apparent difference between the CGXII medium and the other media. These observations demonstrated that the starting d -glucose concentration alone is insufficient to induce isopentenol production in C.
This result is in contrast to E. To examine the role of initial d -glucose concentration for production in CGXII media, we supplemented the medium with a range of starting d -glucose concentrations [0. Under conditions where isopentenol production was detected, C. These results confirm that the starting d -glucose concentration can impact batch-mode production of isopentenol using C. A twofold change in the initial d -glucose concentration can have a fold impact on isopentenol titer. For all experiments, data was generated from three independent biological replicates for each condition, and error bars indicate standard error.
Next, we examined if the initial nitrogen concentration also influenced isopentenol titer. Historically, the carbon:nitrogen ratio C:N ratio is known to impact the detectable levels of metabolites in log phase cultures [ 31 ]. We combined the data for isopentenol production when we varied either nitrogen or d -glucose and re-plotted isopentenol production as a function of the C:N ratio Additional file 3 E. The C:N ratio in M9 media is considerably higher at This ratio may contribute to the differences in measured isopentenol production, since no production in M9 media was observed Additional file 2 A.
Acetate and lactate formation increased under the cultivation conditions we used for isopentenol production Fig. To test if isopentenol titer could be improved by blocking formation of these organic acids, we targeted the genes encoding pyruvate oxidase poxB or lactate dehydrogenase ldhA for deletion. Resulting strains were then transformed with the isopentenol production plasmid. The wild-type C. The rational gene deletions in C.
Rational engineering of C. Analysis of C. Isopentenol titer was measured as described in Additional file 2 A, B. Data was generated from three independent biological replicates for each genotype, and error bars indicate standard error. To examine if isopentenol titers correlate with pathway protein expression, we used selected reaction monitoring SRM based proteomics [ 32 , 33 , 34 ] to assess relative protein levels across these conditions. We observed a similar dependence on the initial d -glucose concentration in CGXII media and a failure to produce isopentenol in M9 or LB irrespective of cultivation format Additional file 4 A.
Crude lysates were prepared for peptide analysis to quantify the five proteins constituting the isopentenol pathway. Pathway proteins from C. This analysis suggests that the failure to produce isopentenol in either LB or M9 media was due to the decreased abundance of the isopentenol pathway proteins. Analysis of HmgR Protein Abundance vs. Isopentenol Titer. Correlation was determined using a linear regression for the Pearson correlation coefficient PCC for the two variables.
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White bars: C. Proteomics data also revealed potential bottlenecks in the isopentenol production pathway. Across the range of cultivation parameters and timepoints tested in wild-type C. To test if higher expression levels of HMGR could improve isopentenol production, we built a set of strains in which the isopentenol production pathway was chromosomally integrated at the idsA locus. To test this process with industrially relevant carbon sources, we demonstrated that C.
We pretreated sorghum biomass using the IL [Ch][Lys] to generate a hydrolysate that contained This hydrolysate also contained 0. When the production strains were grown in the hydrolysate-amended CGXII media, no significant growth defect was observed compared to the growth in pure d -glucose supplemented CGXII media. Substituting hydrolysate for all added water in CGXII media results in a starting concentration of d -glucose at 1. Based on our observations in Fig. In the original hydrolysate-amended condition with 1. When the d -glucose concentration was increased to 3.
Any potential contaminants from IL treated sorghum biomass also did not completely inhibit isopentenol production at these concentrations. These results demonstrate that C. Isopentenol production values are the average of three independent biological replicates, and the error bars represent standard error. The IL concentrations tested are as listed on the x axis.
Emerging Corynebacterium glutamicum systems biology.
Samples were cultivated in well deep well plates. Corynebacterium glutamicum isopentenol production strains were tested for isopentenol production with tolerated levels of ILs refer to Fig. Samples grown in the presence of [C 2 C 1 im][Cl] produced half as much isopentenol as the untreated control Fig. As [Ch][Lys] was inhibitory to C. The reduction of isopentenol titer of these strains largely correlated with the severity of the growth defect, with the exception of [Ch][Cl], which exhibited no impact on doubling time but had a measurable impact on isopentenol production refer to Fig.
In contrast to the cholinium and imidazolium based ILs, one of the protic ILs had modest effects on the production of isopentenol. Taken together, these results indicate that C. HmgR from S. We cloned the S. With the original isopentenol production pathway, C. This improvement in isopentenol production using the pathway variant suggests that the NADH-dependent allele of HmgR was advantageous for isopentenol production in C. An Engineered C. All isopentenol values are the average of biological triplicates, and the error bars represent standard error.
Finally, to complete our process characterization, we assayed isopentenol production from [Ch][Lys] pretreated hydrolysate with the optimized C. These results demonstrate the completion of our microbial bioprocess for isopentenol production in the industrially relevant organism C. Different microbial hosts have innate physiological differences that make them more or less suitable for a final bioconversion process.
However, it is unclear a priori what parameters dictate final product titer of a given heterologous gene pathway. Here we have evaluated the advantages of an industrially relevant microorganism, C. Our highest isopentenol titers with sugars derived from plant biomass are comparable to titers published in E. We also observed that C. Carbon and nitrogen metabolism are closely linked through energy-driven processes such as the generation of ATP. Thus, the carbon to nitrogen ratio C:N is a critical parameter and its effect on growth and production has been reported in variety of microbial hosts [ 42 , 43 ].
This study demonstrates a relationship between initial d -glucose concentrations in CGXII media and isopentenol production, which corresponds to a C:N ratio of 2. A link between starting d -glucose concentration and its impact on gene expression in the context of a heterologous gene pathway has not been described before. This compositional profile indicates that common peptone-based media such as LB or alternatively, defined M9 media were not optimal for our purposes.
Our observations are supported by evidence in E. However, it is still unclear how the initial cell physiology during exponential phase can impact the physiology after d -glucose exhaustion, where much of our batch-mode production occurs. A previous study also examined the impact of C:N ratio on the production of a native molecule [ 45 ], but testing a range of C:N values is not yet common practice when characterizing new products.
The absolute starting d -glucose concentration or C:N ratio may be a general determinant of final product titer and could be beneficial for optimizing other heterologous gene pathways in C. Strain background engineering also played an important role in improving isopentenol titer. Having observed the undesirable formation of acetate and lactic acid in our isopentenol production experiments, we generated new strains to redirect metabolic flux away from these two organic acids.
Genome-scale metabolic flux modeling and analysis [ 47 ] of these strains could reveal unappreciated nuances in host physiology which could inform future metabolic engineering work. Finally, guided by proteomic analysis, pathway engineering resulted in the highest improvements in product titers to the grams per liter level.
A strong correlation between the abundance of HmgR and isopentenol titer suggested HmgR as a potential rate-limiting step in this pathway. While increasing HmgR protein levels improved isopentenol production fourfold, the greatest absolute improvement of isopentenol titer was obtained by utilizing a class II NADH-dependent variant of HmgR from S.
We suggest that additional mutagenesis of HMGR could lead to broadly applicable improvements for other molecules derived from the mevalonate pathway. In addition, technoeconomic analyses [ 48 ] are needed to fully understand the complex variables necessary to implement isopentenol production at an industrial scale. Currently, ILs are valuable and recycled after generating hydrolysates by extensive washing, and thus their concentration in hydrolysate is low [ 36 , 49 ]. However, removal of ILs is not cost-effective in large-scale industrial applications.
As they become less expensive, or used in consolidated one-pot processes [ 50 , 51 ], host strains that can tolerate higher IL concentrations have the potential for an outsized impact on process cost. A higher d -glucose concentration in the hydrolysate could simplify the cultivation process we utilized for producing isopentenol from sorghum biomass hydrolysate. This report describes the successful deployment of a heterologous mevalonate-based pathway in the Gram-positive industrial microorganism, C.
We highlight the intrinsic capability of C. All chemicals and reagents were purchased from Sigma-Aldrich St. Louis, MO or as otherwise indicated, and were of molecular biology grade or higher. When cells were cultivated in a microtiter dish format, plates were sealed with a gas-permeable film Sigma-Aldrich, St. Louis, MO. San Diego, CA. Core primers used to validate genomic deletions are listed in Additional file 7 : Table S2. Plasmids were constructed using chemically competent E.
Integrated gene cassettes and gene deletions were confirmed by colony PCR to verify the complete excision at the targeted open reading frame using and inspected by agarose gel electrophoresis. Whole commercial-grade sorghum plants derived from Sorghum bicolor were grown, harvested, and milled in the harvest cycle by Chromatin Inc New Deal, Texas. Crystalline d -glucose was added to CGXII media with hydrolysate to increase the concentration up to 3.
Corynebacterium glutamicum was made electrocompetent as previously described [ 54 ]. Isopentenol production was analyzed in several different common growth media. To assess the impact of ILs on the growth of C. For the isopentenol toxicity assay, single colonies of C. To measure isopentenol consumption by C. Remaining isopentenol was quantified as described in the following section. Unless otherwise noted, all seed cultures were first inoculated for growth in culture tubes. All strains were cultivated under aerobic growth conditions.
When minimal media was used, cells from a seed culture were sub-cultured twice to adapt cells to growth in the media. After a second back dilution to allow for complete adaptation to growth in minimal media, cultures were then used for growth assays and production in minimal media. The d -glucose concentration was held constant throughout all passaging steps required for adaptation.
Where hydrolysate was used as the carbon source, CGXII media was prepared, substituting the volume used for dH 2 O with the hydrolysate. Crystalline d -glucose was added to this solution to increase the starting d -glucose concentration. To assess isopentenol production under IL stress conditions, the adapted cultures of C. Analytical grade standards were used to calculate analyte concentrations and confirm identification of peaks. Sugars and organic acids were quantified exactly as described in [ 56 ]. Cell lysates were cooled on ice between each round.
The data were acquired using Agilent MassHunter version B.
The Actinobacterium Corynebacterium glutamicum, an Industrial Workhorse.
Acquired SRM data were analyzed by Skyline software version 3. Peptide abundances of the same protein were summed together to assign the protein abundance of a given protein. The average protein value is shown from samples in biological triplicate to assess different timepoints and growth conditions.
Mukhopadhyay A. Tolerance engineering in bacteria for the production of advanced biofuels and chemicals. Trends Microbiol. Engineering tolerance to industrially relevant stress factors in yeast cell factories. Impact of synthetic biology and metabolic engineering on industrial production of fine chemicals. Biotechnol Adv.
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